CN104096225A

CN104096225A - Method for enhancing 13-valent pneumococcal polysaccharide protein bonder immunogenicity

Info

Publication number: CN104096225A
Application number: CN201410199079.5A
Authority: CN
Inventors: 李建平
Original assignee: JIANGSU KANGTAI BIOMEDICAL TECHNOLOGY CO LTD
Current assignee: JIANGSU KANGTAI BIOMEDICAL TECHNOLOGY CO LTD
Priority date: 2014-05-11
Filing date: 2014-05-11
Publication date: 2014-10-15
Anticipated expiration: 2034-05-11
Also published as: CN104096225B

Abstract

The invention discloses a method for enhancing 13-valent pneumococcal polysaccharide protein bonder immunogenicity, and the method is as follows: adding all-powerful epitope peptide (P2) into CRM197A (A chain of diphtheria toxin variant 197), using genetic recombinant Escherichia coli to produce a P2-containing protein carrier P2CRM197A of the A chain of the diphtheria toxin variant 197 (CRM197); and connecting 13 different serotype polysaccharides with the P2-containing protein carrier P2CRM197A by covalent bonds to form a 13-valent pneumococcal polysaccharide-P2CRM197A bonder; compared with a 13-valent pneumococcal polysaccharide-CRM197A bonder obtained by a corresponding protein carrier CRM197A which does not contain the all-powerful epitope peptide (P2), the immunogenicity of the 13-valent pneumococcal polysaccharide-P2CRM197A bonder obtained by the method is increased by 3-5 times than that of a contrast.

Description

One can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates

Technical field

The present invention relates to one and can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates.

Background technology

When polysaccharide is when being covalently linked on protein carrier, haptenic polysaccharide can be transformed into holoantigen, the immunogenicity of polysaccharide is enhanced.The polysaccharide-protein combined vaccine synthetic with the method has been widely used in children's, successfully prevented to comprise the infection of the antibacterials such as streptococcus pneumoniae, epidemic cerebrospinal meningitis coccus and popular haemophilus b type.

Have for the synthesis of the protein carrier of GL-PP conjugate multiple, the popular haemophilus surface protein D producing as tetanus toxoid, diphtheria toxoid, diphtheria toxin, diphtherotoxin variant CRM197 and gene recombination technology etc.; But, due to the immunological characteristic of different albumen, variant with the immunogenicity of the synthetic GL-PP conjugate of different protein carriers, after animal body immunity, for synthesizing by different protein carriers and same polysaccharide the GL-PP conjugate forming, the polysaccharide immunogenic manifesting is also different.As can be seen here, the protein carrier that adopts different technologies to produce, the immunogenicity of the GL-PP conjugate being synthesized is variant.

After entering in animal body, antigen is processed cell (Antigen Process Cell through antigen, APC) process the rear epitope peptide (epitope) [1] that produces, with ajor histocompatibility complex (Major Histocompatibility Complex, MHC) after molecule combination, and then be presented on APC cell surface, and can be identified by T lymphocyte, this is the conclusion that immunology has drawn by experiment.It is now know that, and the immunogenicity of a known epitope peptide depends on three factors:

The first, suitably the generation of epitope peptide;

Second and the presenting of the Major histocompatibility complex molecule of epitope peptide combination;

Presenting of the T cell of the conjugate that three, identification epitope peptide and ajor histocompatibility complex form.

Wherein, lacking of any one link, all will cause immunne response disappearance.

Show with the test that mice carries out, lacking the conjugate molecule that suitable epitope peptide and ajor histocompatibility complex form is the reason that the most often causes animal body immune response disappearance.Ajor histocompatibility complex has the multiform state property of height, and known epitope peptide only just can be bonded to ajor histocompatibility complex by one or several allele (Alleles), instead of whole epitope peptide fragment.In addition, have experimental result to show, process inappropriately due to antigen, or T cell tolerance vacancy, also can cause the disappearance of immune response.

There is experiment to show, the epitope peptide QYIKANSKFIGITEL (being called P2) [2] of tetanus toxin, can with a large amount of different ajor histocompatibility complex Class II combinations, show that it can be by T cell recognition, there is omnipotent immunogenic characteristic, be termed omnipotent epitope peptide.

Omnipotent immunogenicity epitope peptide has homotype and the shaped body combination with multiple mankind's major histocompatibility antigen complex Class II molecules.This omnipotent epitope peptide and the random combination of mankind's major histocompatibility antigen complex Class II molecule can be used for developing synthetic vaccine, because the replying of the acquired immune system of its major part individuality in can activation crowd.

Research shows, P2 epitope peptide forms (QYIKANSKFIGITEL) by the 830-844 aminoacid sequence of tetanus toxin.Enter after body at the albumen that contains this epitope peptide, albumen will be ingested to APC cell, digested degraded.But these epitope peptides will be preserved complete, with ajor histocompatibility complex in conjunction with after, be present in APC cell surface, and by T cell recognition, this show omnipotent epitope peptide in an identical manner with multiple DR molecular action.

MHC molecule is the polygamy receptor of antigen after processing, and its function is in the process of tolerance induction in thymus and periphery immune response exotic antigen, presents the epitope peptide in its antigen.Therefore, MHC molecule must (be allowed better antigen recognition presenting a large amount of epitope peptides, but more consume T cell deposit), and reach balance in a little epitope peptide (a large amount of T cells is laid in, but a little presents exotic antigen effectively).That is to say, if contain in antigen after APC cell is processed and can preserve integrity, and representative a small amount of epitope peptide, with MHC in conjunction with after, just can stimulate a certain amount of T cell, set up immunological memory effect, and this process can not consume excessive T cell, to avoid consuming a large amount of T cells, cause immunologic tolerance.The antigen that contains this epitope peptide has stronger immunogenicity, and this has also just explained why tetanus toxoid has very strong immunogenic reason.

The stimulation epitope peptide of most of T cell of now finding acts on limited for different MHC Class II monomers (haplotype), different animals is preserved and presented different antigen polypeptide regions (epitopes) stimulates its T cell.The gene restriction of this T cell-stimulating activity has hindered with synthetic method and has carried out vaccine development, and this method is for crowds' different on gene application, should be unusual effective method.Those are found to have stimulates the T cytositimulation epitope peptide that multiple mice is individual and/or be associated with most of human body MHC Class II molecules, and the effective way of an omnipotent activating T cell of design is provided.In common proteantigen, add omnipotent epitope peptide (also referred to as omnipotent T cellular antigens bunch) P2, can be by the MHC Class II molecular recognition of most animals.This T cellular antigens bunch can be used in direct inducing T cell, or the B cells produce of offering help is directed to weak immunogenic antibody, the effect of enhancing body Acquired immune response.

Pathogenic bacteria can be expressed high molecular conventionally, and what be wrapped in bacterium surface is capsular polysaccharide, is called for short polysaccharide.For adult, capsular polysaccharide is to have good immunogenicity antigen, can be used for preparing vaccine; But, for children's, 2 years old following infant, it is the non-T of depending on cellular antigens that capsular polysaccharide is considered to.Experiment demonstration, in the time being used as antigen, the response that capsular polysaccharide can be induced wild strain or T cell disappearance mice produces polysaccharide specific IgM antibodies; But, do not induce the conversion of IgM antibody to IgG antibody.Human experimentation also shows, as vaccine, polysaccharide can induce adult to produce protection antibody, but cannot induce the immunne response of infant; That is to say, repeat after immune capsular polysaccharide antigen children's, without the response of antibody enhancing for the second time, T cell memory that also cannot be inducing sustained.

Modern immunological experiment confirms, the advantage of GL-PP combined vaccine and the comparison of holosaccharide vaccine is, the former can induction of immunity response.When the capsular polysaccharide covalent bond of non-dependence T cell be connected to carrier protein and the GL-PP conjugate that forms, in immunity after mammal, can inducing T cell help B cell and produce the IgG antibody that is directed to the polysaccharide part in conjugate.Therefore, GL-PP conjugate induction polysaccharide specific antibody IgM is converted into IgG, the differentiation of memory B cell, and long-standing T cell memory.

Streptococcus pneumoniae, also claim streptococcus pneumoniae, worldwide to cause the mankind to fall ill and one of dead main pathogenic bacteria, especially in infant, chronic heart and lung diseases patient, old people and immunologic hypofunction person, easily cause diseases such as bacterial pneumonia, meningitis, bacteremia and acute otitis media.Estimate the various diseases that has at least every year 1000000 years old following infant and child to die of pneumonia due to coccus according to World Health Organization (WHO) (WHO) report on Epidemiological in 1999.According to industrially developed country, the sickness rate of the invasive pneumococcal pneumonia of 2 years old Infants Below is up to 160 cases in every 100,000 people; In American-European countries, the bacterial meningitis of 25%-40% is caused by streptococcus pneumoniae.The U.S. approximately has 150,000～570,000 routine pneumococcal pneumonias every year, 2600～6200 routine pneumococcal meningitis, and both cause 40,000 people's death altogether every year; The bacteremic sickness rate of streptococcus pneumoniae is in 15,/10 ten thousand～19,/10 ten thousand left and right, case fatality rate approximately 25%～30%.In addition, have 50%～67% for due to streptococcus pneumoniae in bacillary otitis media, be often difficult to radical cure, relapse rate is high, affects child's healthy growth and development.According to statistics, only just go to see a doctor up to seven million peoples time because of otitis media at the annual infant of the U.S., brought heavy burden to medical system.Therefore, when septivalency pneumonia polysaccharide conjugate vaccine is succeeded in developing in U.S.'s Hui Shi pharmacy, and obtain after U.S. FDA production and sales licence, the infectious disease committee of AAP and immunity test consultative committee of the U.S. inoculate this vaccine in the child in 2-4 year who advises immediately infant below 2 years old and hypoimmunity at the beginning of 2000.

Multinomial pneumococcal Epidemiological study result shows, different countries and regions, and pneumococcal popular bacterial strain is that serotype is different.Taking 7 valency pneumococcal Polysaccharide Conjugate Vaccines of Wyeth of the U.S. as example, its vaccine is 80-90% at the coverage rate of North America, and the coverage rate in Europe is about 70-80%, and is only 40-50% at the coverage rate in Asia.Analyze its reason place, the distribution of the streptococcus pneumoniae serotype spectrum of developed country is narrow, and the distribution of the streptococcus pneumoniae serotype spectrum of undeveloped country is broader.Basic reason that why 7 valency pneumococcal Polysaccharide Conjugate Vaccines of Wyeth of the U.S. cannot be applied in Asia that Here it is.According to China and the child in the south east asia 0-5 clinically of nearly 5 years year, the particularly pneumococcal report on Epidemiological of 2 years old Infants Below, find that there is the popular bacterial strain that 13 kinds of serotypes have contained 80-90% substantially, comprise 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F.Accordingly, Pfizer develops 13 valency pneumococcal Polysaccharide Conjugate Vaccines, and the GSK in Europe has developed 10 valency pneumococcal Polysaccharide Conjugate Vaccines.

In the serious infectious disease such as pneumonia that pneumococcal Polysaccharide Conjugate Vaccine is caused by pneumococcal infection at preventing child, meningitis, otitis media, bring into play huge effect.But, on market, the immunogenicity variability of existing pneumococal polysaccharide protein binding vaccine is larger, this variability is except being the reason due to the structural difference of different serotypes polysaccharide, the use of different immunogenic protein carriers is also the immunogen difference main cause that causes 13 valency pneumococal polysaccharide protein conjugates.In some high-risk group, such as children's, old people or immunologic hypofunction people, the immune effect of the proteinpolysaccharide combined vaccine of reduced immunogenicity is not good, and protectiveness is restricted.Therefore, develop the pneumococal polysaccharide protein binding vaccine that immunogenicity is stronger, remain the direction that make great efforts in this field.

Summary of the invention

The object of this invention is to provide one and can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, the omnipotent epi-position peptide protein carrier P2CRM197A that contains that adopts gene recombination technology production introduces omnipotent epitope peptide P2 in proteinpolysaccharide conjugate.Enter after animal body at this GL-PP conjugate, after antigen-presenting cell (APC) is engulfed digestion degraded, its omnipotent epitope peptide P2 and part pneumococal polysaccharide recurring unit, with ajor histocompatibility complex ClassII combination, can effectively stimulate T cell, and then the immunogenicity of pneumococal polysaccharide in enhancing conjugate, cause the antibody concentration that is directed to pneumococcal capsular polysaccharide to increase.

The technical scheme that the present invention takes is as follows:

One can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, it is characterized in that:

Step 1: add omnipotent epitope peptide QYIKANSKFIGITEL (being called for short P2) in the A chain (being called for short CRM197A) of diphtheria toxin, diphtherotoxin variant CRM197, produce the CRM197A protein carrier (being called for short P2CRM197A) that contains P2 by gene recombinaton engineering bacteria;

Step 2: by the pneumococcal capsular polysaccharide of 13 kinds of different serotypes, comprise 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F, be connected to form 13 kinds of unit price pneumococal polysaccharide-P2CRM197A conjugates with P2CRM197A protein carrier respectively by covalent bond;

Step 3: 13 kinds of unit price pneumococal polysaccharide-P2CRM197A conjugates that step 2 is obtained are mixed to get the 13 valency pneumococal polysaccharide protein conjugates that immunogenicity strengthens.

Further, in described P2CRM197A protein carrier, contain X P2,1≤X≤3.

Further, in described P2CRM197A protein carrier, P2 is attached at N-end or the C-end of CRM197A albumen, or is connected to N-end and C-end simultaneously.

Further, between described P2 and CRM197A albumen, be to be connected by GSGSG aminoacid sequence.

Further, described gene recombinaton engineering bacteria is the escherichia coli that build by gene recombination method.

Further, described pneumonia capsular polysaccharide is the capsular polysaccharide that the streptococcus pneumoniae by cultivating respectively 13 different serotypes obtains.

Detailed description of the invention

Illustrate specific embodiment of the invention method below, but be not only confined to following instance.

The first step, the preparation of carrier protein and pneumococal polysaccharide

For effectiveness of the present invention is described, prepare two kinds of carrier proteins, contain the protein carrier P2CRM197A of P2 and not containing the protein carrier CRM197A of P2.Wherein, CRM197A protein carrier is for the synthesis of contrast 13 valency pneumococal polysaccharide-CRM197A conjugate samples.

One, the design of CRM197A protein carrier and P2CRM197A protein carrier aminoacid sequence

1, the design of CRM197A protein carrier aminoacid sequence

Diphtheria toxin, diphtherotoxin is by expressing in diphtheria corynebacterium with the phagus beta of diphtheria toxin, diphtherotoxin gene, and in antibacterial endochylema, existence form is polypeptide, is made up of 560 aminoacid, and molecular weight is 62,000 dalton.Its aminoacid sequence is as follows:

MSRKLFASILIGALLGIGAPPSAHA GADDVVDSSKSFVMENFSSYHGTKPGYV DSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAG GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDG ASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYM AQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

Before secreting to bacterial body, 25 of polypeptide N-end guiding aminoacid sequences cut fall, become by 535 aminoacid and formed, the single chain polypeptide that molecular weight is 58kD is secreted to bacterial body, its aminoacid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWK GFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETI KKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQ AKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

Secreting diphtheria toxin, diphtherotoxin to bacterial body digested is A chain and B chain, is connected therebetween and form a protein molecular by a disulfide bond.These two polypeptide chains have different functions, and A chain is the N-terminal fragment of DT molecule, and molecular weight is 21kD, is made up of 193 aminoacid.A chain is the toxicity funtion part of diphtheria toxin, diphtherotoxin, and it is by eukaryotic cells slurry, by the Isocitrate dehydrogenase (NAD of diphosphonic acid three adenosine ribose (ADP-Ribosyl) ⁺) part is transferred to elongation factor 2 (Elongation Factor-2, EF-2) above, thereby the protein synthesis in inhibition cell, and then cell growth inhibiting, cell injures and deaths caused.B chain is the C-terminal fragment of DT molecule, and molecular weight is approximately 37kD, is made up of 342 aminoacid.The function of B chain is the specific receptor on identification sensitive cells surface, and diphtheria toxin, diphtherotoxin is adsorbed on sensitive cells, helps A chain to enter in cell.

The A chain of diphtheria toxin, diphtherotoxin has good water solublity, and its aminoacid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWK GFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETI KKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQ AKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

[3] are found in research, due to the sudden change of the toxin gene tox on phagus beta, for the not impact that copies of phage; But the toxicity of the toxin being synthesized may disappear, or reduce widely, and form diphtheria toxin mutation (CrossReactingMaterial, CRM), and the serology immunogenicity of diphtheria toxin mutation is still associated with toxin.Such as, diphtheria toxin muton CRM 197, without the toxicity of diphtheria toxin, diphtherotoxin, is due to the 52nd amino acids A chain from amino acid sequence analysis, is mutated into glutamic acid (Glu) by glycine (Gly), its aminoacid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWK EFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETI KKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQ AKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

Experimentation shows, with on other present market for the synthetic protein carrier comparison of pneumococcal conjugated vaccine, diphtheria toxin, diphtherotoxin variant CRM197 protein A chain polypeptide possesses following advantages, as its immunogenicity and diphtheria toxin, diphtherotoxin and diphtheria endotoxin is associated, molecular weight is little, water solublity is high, be easy to produce and carry out macromole synthetic reaction.The clinical use of long-term diphtheria toxoid vaccine, verified its safety and effectiveness.

The design of the P2CRM197A protein carrier aminoacid sequence that 2, contains the omnipotent epitope peptide of P2

Omnipotent epitope peptide P2 is connected on CRM197A protein carrier, constructs a kind of new protein carrier for the synthesis of proteinpolysaccharide conjugate.Omnipotent epitope peptide P2 used can be connected to N-end or the C-end of CRM197A protein carrier; Also two different omnipotent epitope peptides can be connected to respectively to N-end or the C-end of CRM197A protein carrier; Another kind of mode is that two omnipotent epitope peptides self are connected, and then is connected to CRM197A protein carrier N-end or C-end; Also having a kind of mode is that an omnipotent epitope peptide is connected to one end, and two omnipotent epitope peptides that self connect connect the other end.

2-1, the design of P2CRM197A protein carrier aminoacid sequence that contains omnipotent epitope peptide P2

The design of 2-1-1, P2-N-end CRM197A protein carrier (being called P2-CRM197A) aminoacid sequence

By P2 aminoacid sequence QYIKANSKFIGITEL being added to the N-end of CRM197A protein carrier, form a new albumen, its aminoacid sequence is as follows:

QYIKANSKFIGITELGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDW

KEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGT

EEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

Between the N-end of P2 aminoacid sequence and CRM197A protein carrier, inserted GSGSG fragment and be connected, the albumen building with the method is called P2-CRM197A protein carrier.

The design of 2-1-2, CRM197AC-end-P2 protein carrier (being called CRM197A-P2) aminoacid sequence

By P2 aminoacid sequence QYIKANSKFIGITEL being added to the C-end of CRM197A protein carrier, form another kind of new albumen, its aminoacid sequence is as follows:

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG QYIKANSKFIGITEL

Between the C ?end of P2 aminoacid sequence and CRM197A protein carrier, inserted GSGSG fragment and be connected, the albumen building with the method is called CRM197A ?P2 protein carrier.

The design of 2-1-3, P2-N-end CRM197AC-end-P2 protein carrier (being called P2-CRM197A-P2) aminoacid sequence

By two P2 aminoacid sequence QYIKANSKFIGITEL being added to respectively to N-end and the C-end of CRM197A protein carrier, form a kind of new albumen, its aminoacid sequence is as follows:

QYIKANSKFIGITELGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG QYIKANSKFIGITEL

The N of P2 aminoacid sequence and CRM197A protein carrier ?end and C ?inserted GSGSG fragment between end and connected, the albumen building with the method be called P2 ?CRM197A ?P2 protein carrier.

The design of 2-1-4, P2P2-N-end CRM197A protein carrier (being called P2-P2-CRM197A) aminoacid sequence

By two P2 aminoacid sequence QYIKANSKFIGITEL self are connected, and then be added to the N-end of CRM197A protein carrier, form a kind of new albumen, its aminoacid sequence is as follows:

QYIKANSKFIGITELGSGSG QYIKANSKFIGITELGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

Between two P2 aminoacid sequences that self connect, and with the N ?end of CRM197A protein carrier between inserted GSGSG fragment and be connected, the albumen building with the method is called P2 ?P2 ?CRM197A protein carrier.

The design of 2-1-5, CRM197AC-end-P2P2 protein carrier (being called CRM197A-P2-P2) aminoacid sequence

By two P2 aminoacid sequence QYIKANSKFIGITEL self are connected, and then be added to the C-end of CRM197A protein carrier, form a kind of new albumen, its aminoacid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG QYIKANSKFIGITELGSGSG QYIKANSKFIGITEL

Self connect between P2 aminoacid sequence at two, and with the C ?end of CRM197A protein carrier between inserted GSGSG fragment and be connected, the albumen building with the method is called CRM197A ?P2 ?P2 protein carrier.

2 ?1 ?6, P2P2 ?N ?end CRM197AC ?Mo Duan ?P2 protein carrier (be called P2 ?P2 ?CRM197A ?P2) design of aminoacid sequence

By two P2 aminoacid sequence QYIKANSKFIGITEL self are connected, and then be added to the N-end of CRM197A protein carrier; In addition, a P2 is added to the C-end of CRM197A protein carrier, forms another kind of new albumen, its aminoacid sequence is as follows:

QYIKANSKFIGITELGSGSG QYIKANSKFIGITELGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG QYIKANSKFIGITEL

Self connect between P2 aminoacid sequence at two, and with the C ?end of CRM197A albumen between inserted GSGSG fragment and be connected, the albumen building with the method is called P2 ?P2 ?CRM197A ?P2 protein carrier.

2 ?1 ?7, P2 ?N ?end CRM197AC ?Mo Duan ?P2P2 protein carrier (be called P2 ?CRM197A ?P2 ?P2) design of aminoacid sequence

By a P2 aminoacid sequence QYIKANSKFIGITEL being connected to the N-end of CRM197A protein carrier; In addition, two P2 are carried out to self and connect, and then be added to the C-end of CRM197A protein carrier, form another kind of new albumen, its aminoacid sequence is as follows:

QYIKANSKFIGITELGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG QYIKANSKFIGITELGSGSG QYIKANSKFIGITEL

Self connect between P2 aminoacid sequence at two, and with the C-end of CRM197A protein carrier between inserted GSGSG fragment and be connected, the albumen building with the method is called P2-CRM197A-P2-P2 protein carrier.

Two, the structure of CRM197A protein carrier and the P2CRM197A protein carrier expression plasmid that contains omnipotent epitope peptide

1, the structure of CRM197A protein carrier expression plasmid

Obtain CRM197 albumen complete amino acid sequence PRF:224021 from GenBank, determine the A chain fragment of CRM197 albumen, the A chain fragment that amino acid/11-193 of CRM197 are CRM197.On this basis, the amino acid whose nucleotide sequence of this fragment is optimized, so as in escherichia expression system high efficient expression.Adopt homemade expression plasmid, with NdeI enzyme identification plasmid site CATATG, BamHI enzyme recognition site GGATCC.Through the gene order of CRM197A is analyzed, in sequence, without NdeI and BamHI restriction enzyme site.CRM197A protein gene composition sequence is as follows:

CATATG

GGTGCGGACG?ACGTTGTGGA?CTCCTCAAAA?TCGTTTGTCATGGAAAACTT?CAGCTCTTAT

CATGGCACCA?AACCGGGTTA?CGTGGACTCC?ATTCAGAAGGGCATCCAAAA?ACCGAAGTCA

GGCACCCAGG?GTAACTACGA?TGACGATTGG?AAG GAATTCTACAGCACGGA?CAATAAGTAT

GATGCGGCCG?GCTACTCTGT?TGACAACGAA?AATCCGCTGAGTGGTAAAGC?AGGCGGTGTG

GTTAAGGTCA?CCTATCCGGG?TCTGACGAAA?GTTCTGGCGCTGAAGGTCGA?TAACGCCGAA

ACCATTAAAA?AGGAACTGGG?CCTGTCTCTG?ACCGAACCGCTGATGGAACA?AGTGGGTACG

GAAGAATTTA?TCAAACGTTT?CGGCGATGGT?GCATCGCGTGTCGTGCTGAG?CCTGCCGTTT

GCTGAAGGCA?GTTCCTCAGT?GGAATACATT?AACAATTGGGAACAAGCAAA?AGCTCTGTCA

GTTGAACTGG?AAATCAATTT?CGAAACGCGT?GGCAAACGCGGTCAAGATGC?TATGTATGAA

TATATGGCTC?AGGCGTGTGC?GGGCAATCGC?GTCCGTCGCT?AA

GGATCC

By in the PCR product of blank plasmid and synthetic CRM197A protein gene, add respectively NdeI enzyme and BamHI enzyme to carry out double digestion reaction; After purification, in linked system, add T4 ligase to connect, purification expression plasmid after completing, and identify with PCR identification system and restriction enzyme mapping.With BL21 (DE3) competent cell, expression plasmid is converted in cell to screening and cloning.Obtain after positive expression engineering bacteria, set up seed bank, comprise main seed and work seed, and be stored in-20 DEG C of following refrigerators.

The structure of the P2CRM197A protein carrier expression plasmid that 2, contains omnipotent epitope peptide

The structure of 2-1, P2-CRM197A protein carrier expression plasmid

Adopt homemade blank expression plasmid, with NdeI enzyme identification plasmid site CATATG, BamHI enzyme recognition site GGATCC.Through P2CRM197A protein carrier gene order is analyzed, in sequence, without NdeI and BamHI restriction enzyme site.The synthetic complete sequence of P2-CRM197A gene is as follows:

CATATG

CAATACATCA?AGGCGAACAG?CAAATTCATC?GGCATCACGGAACTGGGCTC?GGGCTCTGGC

GTGCGGACG?ACGTTGTGGA?CTCCTCAAAA?TCGTTTGTCATGGAAAACTT?CAGCTCTTAT

ATGGCACCA?AACCGGGTTA?CGTGGACTCC?ATTCAGAAGGGCATCCAAAA?ACCGAAGTCA

GGCACCCAGG?GTAACTACGA?TGACGATTGG?AAG GAATTCTACAGCACGGA?CAATAAGTAT

GATGCGGCCG?GCTACTCTGT?TGACAACGAA?AATCCGCTGAGTGGTAAAGC?AGGCGGTGTG

GTTAAGGTCA?CCTATCCGGG?TCTGACGAAA?GTTCTGGCGCTGAAGGTCGA?TAACGCCGAA

ACCATTAAAA?AGGAACTGGG?CCTGTCTCTG?ACCGAACCGCTGATGGAACA?AGTGGGTACG

GAAGAATTTA?TCAAACGTTT?CGGCGATGGT?GCATCGCGTGTCGTGCTGAG?CCTGCCGTTT

GCTGAAGGCA?GTTCCTCAGT?GGAATACATT?AACAATTGGGAACAAGCAAA?AGCTCTGTCA

GTTGAACTGG?AAATCAATTT?CGAAACGCGT?GGCAAACGCGGTCAAGATGC?TATGTATGAA

TATATGGCTC?AGGCGTGTGC?GGGCAATCGC?GTCCGTCGCT?AA

GGATCC

By in the PCR product of blank plasmid and synthetic P2CRM197A protein gene, add respectively NdeI enzyme and BamHI enzyme to carry out double digestion reaction; After purification, in linked system, add T4 ligase to connect, purification expression plasmid after completing, and identify with PCR identification system and restriction enzyme mapping.With BL21 (DE3) competent cell, expression plasmid is converted in cell, screening and cloning, and identify with PCR identification system and restriction enzyme mapping.Obtain after positive expression engineering bacteria, set up seed bank, comprise main seed and work seed, and be stored in-20 DEG C of following refrigerators.

The structure of 2-2, P2-CRM197A-P2 protein carrier expression plasmid

Adopt homemade expression plasmid, with NdeI enzyme identification plasmid site CATATG, BamHI enzyme recognition site GGATCC.Through P2-CRM197A-P2 protein gene sequence is analyzed, in sequence, without NdeI and BamHI restriction enzyme site.The synthetic complete sequence of P2CRM197AP2 gene is as follows:

CATATG

CAATACATCA?AGGCGAACAG?CAAATTCATC?GGCATCACGGAACTGGGCTC?GGGCTCTGGC

GTGCGGACG?ACGTTGTGGA?CTCCTCAAAA?TCGTTTGTCATGGAAAACTT?CAGCTCTTAT

ATGGCACCA?AACCGGGTTA?CGTGGACTCC?ATTCAGAAGGGCATCCAAAA?ACCGAAGTCA

GGCACCCAGG?GTAACTACGA?TGACGATTGG?AAG GAATTCTACAGCACGGA?CAATAAGTAT

GATGCGGCCG?GCTACTCTGT?TGACAACGAA?AATCCGCTGAGTGGTAAAGC?AGGCGGTGTG

GTTAAGGTCA?CCTATCCGGG?TCTGACGAAA?GTTCTGGCGCTGAAGGTCGA?TAACGCCGAA

ACCATTAAAA?AGGAACTGGG?CCTGTCTCTG?ACCGAACCGCTGATGGAACA?AGTGGGTACG

GAAGAATTTA?TCAAACGTTT?CGGCGATGGT?GCATCGCGTGTCGTGCTGAG?CCTGCCGTTT

GCTGAAGGCA?GTTCCTCAGT?GGAATACATT?AACAATTGGGAACAAGCAAA?AGCTCTGTCA

GTTGAACTGG?AAATCAATTT?CGAAACGCGT?GGCAAACGCGGTCAAGATGC?TATGTATGAA

TATATGGCTC?AGGCGTGTGC?GGGCAATCGC?GTCCGTCGCTAAGGCTCGGG?CTCTGGCCAA

TACATCAAGG?CGAACAGCAA?ATTCATCGGC?ATCACGGAAC?TG

GGATCC

By in the PCR product of blank plasmid and synthetic P2-CRM197A-P2 gene, add respectively NdeI enzyme and BamHI enzyme to carry out double digestion reaction; After purification, in linked system, add T4 ligase to connect, purification expression plasmid after completing, and identify with PCR identification system and restriction enzyme mapping.With BL21 (DE3) competent cell, expression plasmid is converted in cell, screening and cloning, and identify with PCR identification system and restriction enzyme mapping.Obtain after positive expression engineering bacteria, set up seed bank, comprise main seed and work seed, and be stored in-20 DEG C of following refrigerators.

The structure of 2-3, CRM197A-P2, P2-P2-CRM197A, CRM197A-P2-P2, P2-P2-CRM197A-P2 and P2-CRM197A-P2-P2 protein carrier expression plasmid

Method and upper joint " structure of 2-1, P2-CRM197A protein carrier expression plasmid " are identical.

Three, the preparation that contains omnipotent epitope peptide P2CRM197A protein carrier and CRM197A protein carrier

Experimental result of the present invention shows, CRM197A protein carrier is similar with the characteristic of the CRM197A protein carrier that contains omnipotent epitope peptide; Therefore, the purification process of these protein carriers is similar, below taking the CRM197A protein carrier that contains omnipotent epitope peptide as example illustration method.

1, express the preparation that contains omnipotent epitope peptide P2CRM197A protein carrier engineering bacteria

The plasmid of each expressing protein carrier is proceeded to competent cell by standard molecular biological method, and express calibrating.Filter out expressing quantity high, and by the clone with antiserum assay approval, set up main seed bank and work seed bank.

2, contain omnipotent epitope peptide P2CRM197A protein carrier and express the fermentation of engineering bacteria

From colibacillus engineering work seed bank cryogenic refrigerator, take out a work seed pipe that contains omnipotent epitope peptide P2CRM197A protein carrier, at room temperature thaw; By in aseptic the bacterium liquid in the seed pipe culture medium that is transferred to 50 milliliters, at 37 DEG C, shake in the shaking table of speed for 180rpm and be cultured to OD ₆₀₀=1.0 left and right; By in the culture medium of bacterium liquid aseptic inoculation to 1 liter, at 37 DEG C, in the shaking table that to shake speed be 180rpm, be cultured to OD ₆₀₀=1.0 left and right; Inoculation seed liquor to 50 rises in 20 liters of culture medium in fermentation tank, at 37 DEG C, under 240rpm condition, ferments, and works as OD ₆₀₀during to 7-8, add IPTG induction recombiant protein synthetic in antibacterial; Fermentation, after 14 hours, stops fermentation, centrifugal, collects thalline stand-by.

The purification of the P2CRM197A protein carrier that 3, contains omnipotent epitope peptide

The protein carrier that contains different omnipotent epitope peptides due to structure is all taking CRM197A as main body, experiment shows, although added omnipotent epitope peptide, but the parameter influence to protein purification is little, only need on the technological parameter of purification CRM197A protein carrier, carry out some and modify, just can set up the purification process of other CRM197A protein carriers that contain omnipotent epitope peptide.

The 50g wet thallus of weighing rises in Centrifuge Cup in 2-, adds 300ml1xPBSpH7.0 buffer suspendible thalline, stirring and evenly mixing 30min on magnetic stirring apparatus; At 4 DEG C, 4000rpm, centrifugal 20min, abandons supernatant, collects thalline; Repeat this step twice; Add 300ml1xPBSpH7.0 to thalline centrifuge tube, on homogenizer, carry out fragmentation; At 4 DEG C, 10000rpm, centrifugal 20min; Collecting precipitation, abandons supernatant; Add 300ml1xPBSpH7.0 buffer, on magnetic stirring apparatus, stir 30min; At 4 DEG C, 4000rpm, centrifugal 20min; Abandon supernatant, collect inclusion body, add the inclusion body after 900ml degeneration buffer extremely washs, at 25 DEG C, the centrifugal 30min of 10000rpm, collects supernatant, abandons precipitation; Centrifuged supernatant is transferred in 6-8KD bag filter to sealing bag filter; Put bag filter in 10 liters of renaturation buffers 1, under room temperature, on magnetic stirring apparatus, stir dialysed overnight; Proceed to bag filter in 10 liters of renaturation buffers 2 next day, stirring at room temperature dialysis 8-10 hour; Bag filter is proceeded in 10 liters of renaturation buffers 3 to stirring at room temperature dialysed overnight; Proceed to bag filter in 10 liters of renaturation buffers 4 next day, stirring at room temperature dialysis 8-10 hour; Bag filter is proceeded in 10 liters of renaturation buffers 5 to stirring at room temperature dialysed overnight; Proceed to bag filter in 2 liters of deposit buffer next day, stirring at room temperature dialysis 8-10 hour; Change deposit buffer secondary, room temperature dialysed overnight; Get 1ml dialysis solution, the centrifugal 10min of room temperature 12000rpm, collects supernatant, surveys protein concentration; DEAE glue post by protein solution loading to pre-balance, with Gradient elution, and collects destination protein peak; Then loading to Phenyl drainage column is further purified, and collects eluting peak; Last loading SP glue post, collects eluting peak; By collect obtain purification destination protein go in bag filter, in the sodium chloride of 0.15M, dialyse, after completing, be transferred at 4 DEG C, store stand-by.

Four, the preparation of bacterial capsule polysaccharide

The present invention is to pneumococcal 13 kinds of serotype pneumococcal capsular polysaccharides, comprise 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F, carry out purification, for use in synthesizing of polysaccharide conjugate, its quality reaches the polysaccharide quality standard of the synthetic GL-PP combined vaccine of WHO.

1, the foundation of streptococcus pneumoniae seed bank

13 serotype streptococcus pneumoniae buying from ATCC, 9163), 3 (article No.s: 10813), 4 (article No.s: BAA-334), 5 (article No.s: BAA-341), 6A (article No.: BAA-659), 6B (article No.: 700675), 7F (article No.: 10351), 9V (article No.: 700671), 14 (article No.s: 6314), 18C (article No.: 10356), 19A (article No.: 700673), 19F (article No.: 700905) and 23F (article No.: 700669) (the article No.: that comprises 1.Take out the strain (primordial seed is criticized) that ATCC buys, add the streptococcus pneumoniae AHC fluid medium of 0.5ml that strain is mixed, get 0.25ml bacterium liquid in 5% Sanguis caprae seu ovis AHC culture fluid.Postvaccinal 5% Sanguis caprae seu ovis AHC culture fluid pipe is placed on the cultivation shaking table of 36 DEG C ± 1 DEG C, shakes fast 120rpm, cultivates 12-20 hour.Treat OD ₆₀₀to 1.0 o'clock, 5% Sanguis caprae seu ovis AHC culture fluid is seeded on AHC agar culture plate with inoculating loop, be placed in the incubator of 36 DEG C ± 1 DEG C and cultivate 12-20 hour.With inoculating loop, 1 on AHC agar plate, in the AHC culture fluid to several colony inoculations in 10ml, is placed in to 36 DEG C ± 1 DEG C, on cultivation shaking table, cultivates 12 hours, shake fast 150-200rpm.Antibacterial OD in culture fluid ₆₀₀grow to 1.0, take out 5ml antibacterial AHC culture fluid and be inoculated in the fresh AHC culture fluid of 200ml, be placed on the cultivation shaking table of 36 DEG C ± 1 DEG C and cultivate about 12 hours, shake fast 150-200rpm.OD ₆₀₀to 1.0 o'clock, inoculum is sub-packed in 200 small test tubes to centrifugal (4000rpm with 1ml, 10min), remove supernatant culture fluid, then add the fresh AHC culture fluid of 0.5ml and the germfree defatted milk of 0.5ml, mix quick freezing on ethanol dry ice.Vacuum freeze-drying, numbering, is stored in 4 DEG C of refrigerators as main seed lot.Take out main seed lot and set up, according to main seed method for building up, inoculum is inoculated in the fresh AHC culture fluid of another 200ml, be placed on the cultivation shaking table of 36 DEG C ± 1 DEG C and cultivate about 12 hours, shake fast 150-200rpm.Treat OD ₆₀₀to 1.0 o'clock, inoculum is sub-packed in 200 small test tubes with 1ml, centrifugal (4000rpm, 10min), removes supernatant culture fluid, then adds the fresh AHC culture fluid of 0.6ml and 40% glycerite of 0.4ml, mixes.Quick-freezing, on dry ice, is stored in-70 DEG C of cryogenic refrigerators as work seed.

2, pneumococcal fermentation

From seed bank, take out lyophilizing work seed pipe, add 1mlAHC enrichment culture liquid to dissolve lyophilised bacteria.The bacterium liquid of dissolving is inoculated in 5mlAHC enrichment culture liquid test tube, at CO ₂in incubator, leave standstill overnight incubation.When observation has bacterial growth, bacterium liquid is inoculated in the AHC enrichment culture liquid triangular flask of 100ml.Put culture bottle in shaking table, at 36 DEG C, 200rpm/min is cultured to OD ₆₀₀be 1.0.100ml inoculum is inoculated in respectively to 2 to be equipped with in the AHC enrichment culture liquid bottle of 1 liter.Put culture bottle in shaking table, at 36 DEG C, 200rpm/min is cultured to OD ₆₀₀be 1.0.The aseptic filtration AHC enrichment culture liquid of 35 liters is injected to 50 liters of fermentation tanks.Inoculate bacterium liquid that 2 liters of OD are 1 to fermentation tank.When bacterial growth arrives plateau, sterilization, results culture supernatant.

3, the purification of capsular polysaccharide

Use deep layer membrane filtration, further remove remaining antibacterial and fragment.By aseptic supernatant culture fluid 100Kd ten times of ultrafiltration and concentration of ultrafilter membrane (about 600ml).Carry out ultrafiltration cleaning with 6 liters of 25mM sodium acetate solution.Adding HB storage liquid to make ultimate density is 1% (w/v), mixes, and solution is positioned over to freezer and spends the night.Centrifugal, 4000rpm, 1 hour, collect polysaccharide/HB precipitation, abandon centrifugal liquid.Add the sodium acetate+1%HB of 25mM to polysaccharide/HB precipitation vessel, stirring suspension precipitation, centrifugal, 4000rpm, 1 hour, collect polysaccharide/HB precipitation, abandon centrifugal liquid.Repeat 3 times this step.With the 0.25M sodium chloride solution dissolution precipitation of 600ml.Centrifugal, 4000rpm, 1 hour, abandons insoluble contaminant nucleic acid.Add 10% liquor kalii iodide to enter in polysaccharide+HB mixed solution, mix, potassium iodide ultimate density is 0.5%, solution is placed in to freezer and spends the night.Use deep bed filter filtering solution, remove the HB/I precipitation in solution, and by the precipitation on 0.25M sodium chloride/0.5% liquor kalii iodide cleaning deep bed filter, collect filtrate, discard precipitation.Rough polysaccharide solution is passed into active carbon deep bed filter and carry out circulating filtration 30 minutes (4% active carbon/0.5mg/ml polysaccharide crude solution).Add sodium phosphate buffer pH6.8, ultimate density 25mM in polysaccharide solution.Above solution is crossed to HA post (50-100ml), circulate 30 minutes.Wash a post 4-5 column volume with identical phosphate solution.With the concentrated polysaccharide solution 5-of 30Kd membrane ultrafiltration doubly, use without heat source water ultrafiltration and clean polysaccharide solution.With 0.22 μ m membrane filtration, lyophilizing.

Second step: the preparation of 13 kinds of serotype unit price pneumococal polysaccharide-P2CRM197A conjugates

In the chemical constitution of different streptococcus pneumoniae serotype capsular polysaccharides, contain different groups, need to adopt different synthetic methods by polysaccharide covalent key be connected to protein carrier and form conjugate, and output and the immunogenicity of the synthetic conjugate of distinct methods are different.The present invention is according to experimental result, adopt three kinds of synthetic methods, reduce amine method, CDAP method (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride method) and ADH method (adipyl dihydrazide method), synthesize specific GL-PP conjugate.

One, 13 kinds of streptococcus pneumoniae serotype capsular polysaccharide-P2-CRM197A conjugates is synthetic

1, streptococcus pneumoniae serotype 1 capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

Take the Pn1 degradation of polysaccharide of 5mg in reaction bulb, the 1mol/LNaCl that measures 0.5ml adds in reaction bulb; Magnetic agitation is dissolved polysaccharide completely.Record the initial pH of polysaccharide solution, measure respectively appropriate CDAP solution, add in reaction bulb.Stirring reaction 1.5min under room temperature, measures the pH of solution when 30s.After 1.5min, with the pH to 9.5 of 0.2mol/LNaOH regulator solution, stirring reaction 3min under room temperature (maintaining pH 9.5 with 0.2mol/LNaOH).After 3min immediately to the P2-CRM197A albumen that adds 5mg in reaction bulb, at room temperature (25 DEG C) stirring reaction 1h.Measure 37.5 μ l2mol/L lysines and add in reaction bulb, with 0.1NHCl regulator solution pH to 9.0.Stirring reaction 30min under room temperature, transfers to reaction at 4 DEG C by reaction bulb and spends the night.Reactant mixture is transferred in bag filter (MWCO6-8000), at 4 DEG C, to 0.85%NaCl solution dialysis 3 times, 6L/ time.After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt the polysaccharide conjugate after the dialysis of SepharoseCL-4B gel column purification, and collect conjugate peak, sampling censorship.

2, streptococcus pneumoniae serotype 3 capsular polysaccharides-P2-CRM197A protein conjugates is synthetic

Take the Pn3 degradation of polysaccharide of 20mg in reaction bulb, the 0.15MNaCl that measures 2ml adds in reaction bulb; Magnetic agitation is dissolved polysaccharide completely.Measure appropriate CDAP solution, add in reaction bulb.Stirring reaction 1.5min under room temperature, measures the pH of solution when 30s.After 1.5min, with the pH to 9.5 of 0.2mol/LNaOH regulator solution, stirring reaction 3min under room temperature (maintaining pH 9.5 with 0.2mol/LNaOH).Add ultimate density be the ADH of 0.8M to reaction bulb, stirring and evenly mixing, reacts under room temperature 2 hours.Polysaccharide after derivation is gone in the bag filter of 10KD 0.15MNaCl solution is dialysed, change liquid three times.Loading G-50 post, uses 0.15MNaCl eluting, collects void volume peak.Then be transferred in bag filter, to water dialysis, change liquid three times.Take the Pn3 polysaccharide after 5mg derivation, be dissolved in the 0.15MNaCl solution of 0.5ml, add the P2-CRM197A albumen of 5mg, after stirring and evenly mixing, add the EDC of 30mM, at room temperature react 4 hours, transfer to reaction at 4 DEG C and spend the night.Reactant mixture is transferred in bag filter (MWCO6-8000), at 4 DEG C, to 0.85%NaCl solution dialysis 3 times, 6L/ time.After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt the polysaccharide conjugate after the dialysis of SepharoseCL-4B gel column purification, and collect conjugate peak, sampling censorship.

3, streptococcus pneumoniae serotype 4 capsular polysaccharides-P2-CRM197A protein conjugates is synthetic

Take 5mg activated polysaccharide to reaction bulb, measure the 0.5M sodium phosphate buffer of 100 μ l, add in reaction bulb, measure the P2-CRM197A albumen of 5mg to reaction bulb, magnetic agitation is dissolved polysaccharide completely; Measure 0.5ml pure water and add in reaction bulb, magnetic agitation mixes; The sodium cyanoborohydride that takes 5.0mg, adds in reaction bulb.The dry bath that reaction system is placed in to 30 DEG C reacts 12h.After reaction finishes, the 0.15M sodium chloride solution that measures 1.5ml adds in reaction bulb.The sodium borohydride that takes 2.5mg, adds in reaction bulb.Reaction system is placed at 22 DEG C reacts 5h; Reactant mixture is transferred to bag filter (MWCO12-14Kd), at 4 DEG C, to 0.15Msodium chloride solution dialysis 3 times, the liquid measure of dialysing 6L at every turn; After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt the polysaccharide conjugate after the dialysis of SepharoseCL-4B gel column purification, and collect conjugate peak, sampling censorship.

4, streptococcus pneumoniae serotype 5 capsular polysaccharides-P2-CRM197A protein conjugates is synthetic

Take 5.0mg activated polysaccharide, be added in reaction bulb, to the 0.5M sodium phosphate buffer that adds 100 μ l in reaction bulb; The P2-CRM197A albumen that takes 4.0mg, adds in reaction bulb, measures 0.5ml pure water and adds in reaction bulb, and magnetic agitation makes reactants dissolved, the pH of assaying reaction system; Take 5.0mg sodium cyanoborohydride, add in reaction bulb; Reaction system is placed under room temperature and is reacted 48 hours; Take the sodium borohydride of 2.5mg, be dissolved in 10 μ l pure water, after mixing and dissolve completely with liquid-transfering gun, add in reaction bulb; Reaction system is placed at 23 DEG C to stirring reaction 5 hours; Reactant mixture is transferred in bag filter (MWCO6-8KD), at 4 DEG C, to 0.15M sodium chloride solution dialysis 3 times, within every 5 hours, changes liquid once; After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt the polysaccharide conjugate after the dialysis of Sepharose CL-4B gel column purification, and collect conjugate peak, sampling censorship.

5, streptococcus pneumoniae serotype 6A capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

Take 6.0mg activation Pn6A polysaccharide, be dissolved in 1mL purified water, be stirred to and dissolve the rear original ph of surveying completely; With 0.1MNaOH adjusting reacting liquid pH value to 7.0; To the P2-CRM197A albumen that adds 4mg in reaction system, stirring and evenly mixing; Take the sodium cyanoborohydride of 5.0mg, add in above-mentioned reaction bulb, room temperature reaction 18 hours; Reaction finishes rear sampling censorship; Take the Sodium borohydride of 2.7mg, add in above-mentioned reaction bulb, room temperature reaction 5 hours; Reaction finishes rear sampling censorship; Reactant mixture is transferred to bag filter, at 4 DEG C, to 0.15M sodium chloride solution dialysis 5 times, 6L/ time.After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt the polysaccharide conjugate after the dialysis of SepharoseCL-4B gel column purification, and collect conjugate peak, sampling censorship.

6, streptococcus pneumoniae serotype 6B capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

The Pn6B that takes 5.0mg is dissolved in 1mL purified water, is stirred to and dissolves the rear original ph of surveying completely; With 0.1MNaOH adjusting reacting liquid pH value to 7.0; To the P2-CRM197A albumen that adds 2.5mg in reaction system, stirring and evenly mixing; Take the sodium cyanoborohydride of 5.0mg, add in above-mentioned reaction bulb, room temperature reaction 20 hours; Take the Sodium borohydride of 2.5mg, add in above-mentioned reaction bulb, room temperature reaction 6 hours; Reactant mixture is transferred to bag filter, at 4 DEG C, to 0.15MNaCl solution dialysis 5 times, 6L/ time; After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt the polysaccharide conjugate after the dialysis of SepharoseCL-4B gel column purification, and collect conjugate peak, sampling censorship.

7, streptococcus pneumoniae serotype 7F capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

The Pn7F polysaccharide that takes 10.0mg, is dissolved in 1mL purified water, is stirred to completely and dissolves; Drip respectively 0.1MNaOH solution to polysaccharide solution, regulate pH to 7.0; To the P2-CRM197A albumen that adds 3.5mg in reaction system, stirring and evenly mixing; Take the sodium cyanoborohydride of 5.0mg, add in above-mentioned reaction bulb, room temperature reaction 20 hours; In reaction bulb, add 990 μ l pure water, stirring and evenly mixing; Take the Sodium borohydride of 2.5mg, add in above-mentioned reaction bulb, room temperature reaction 6 hours; Reactant mixture is transferred to bag filter (MWCO6000-8000), at 4 DEG C, to 5mM succinate/0.9% sodium chloride buffer dialysis 5 times, 6L/ time; After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt the polysaccharide conjugate after the dialysis of Sepharose CL-4B gel column purification, and collect conjugate peak, sampling censorship.

8, streptococcus pneumoniae serotype 9V capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

Take the Pn9V activated polysaccharide of 10mg, the sodium phosphate buffer, the 125 μ l pure water that measure 125 μ l add in reaction bulb, and magnetic agitation is dissolved polysaccharide completely; Measure the P2-CRM197A albumen of 15mg to adding in reaction bulb, stirring and dissolving is complete; Take the NaBH of 10mg ₃(CN), add in reaction bulb; Reaction system is placed at 22 DEG C and is reacted 48 hours; Take the NaBH of 2.5mg ₄, add in reaction bulb, react 5 hours at 22 DEG C; By reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt AKTA system, the polysaccharide conjugate after the dialysis of SepharoseCL-4B gel column purification, and collect in conjunction with peak.

9, streptococcus pneumoniae serotype 14 capsular polysaccharides-P2-CRM197A protein conjugates is synthetic

Take the Pn14 activated polysaccharide of 5mg, measure the P2-CRM197A albumen of 1ml3.9mg, add in reaction bulb, magnetic agitation is dissolved polysaccharide completely; Add after weak reductant sodium cyanoborohydride5mg, at 22 DEG C, react 48 hours; Add strong reductant sodium borohydride2.5mg, under room temperature, react 4 hours; Reaction mixture is transferred in bag filter (MWCO12-14KD), with the dialysis solution rinse reaction bulb of 2ml; At 4 DEG C, to 0.15M sodium chloride solution dialysis 3 times, 6L/ time, within every 5 hours, change liquid once.After dialysis finishes, collect dialysis solution, 10000rpm, gets supernatant after centrifugal 10min, adopts the polysaccharide conjugate after the dialysis of Sepharose CL-4B gel column purification, and collects conjugate peak.

10, streptococcus pneumoniae serotype 18C capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

Take the Pn18C degradation of polysaccharide of 5mg, with the dissolving of 1mL1M sodium chloride solution; After dissolving completely, survey its original ph; Add appropriate CDAP solution, under room temperature, stir 1.5min, add the pH to 9.0 of 0.2M NaOH solution regulator solution, afterwards in room temperature reaction 3min; The P2-CRM197A albumen that adds 10mg reacts 45min at 25 DEG C; After reaction finishes, add 37.5 μ L2M lysine solutions; At 25 DEG C, reacting 30min is placed on 4 DEG C of reactions and spends the night; Reaction mixture is gone in bag filter (MWCO6000-8000), at 4 DEG C, to 0.85% sodium chloride solution dialysis, change liquid 3 times.6L/ time, within every 5 hours, change liquid once.After dialysis finishes, collect dialysis solution, the centrifugal 10min of 10000rpm, gets supernatant, employing CL-4B gel-purified this in conjunction with polysaccharide, and collect conjugate peak; Detect albumen and polyoses content in conjugate solution.

11, streptococcus pneumoniae serotype 19A capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

Take 10.0mg oxidation Pn19A polysaccharide, be dissolved in the buffer of 0.5mL, put bar magnet in reaction bulb, be stirred to polysaccharide and dissolve completely; Add the P2-CRM197A albumen of 10mg, stirring and evenly mixing; The sodium cyanoborohydride that takes 5.0mg, is added in reaction bulb, at room temperature reacts 20 hours; The sodium borohydride that takes 2.5mg, adds in reaction bulb, at room temperature reacts 5 hours; Reaction mixture is gone in bag filter (MWCO6000-8000), at 4 DEG C, to 0.85% sodium chloride solution dialysis, change liquid 3 times, 6L/ time, within every 5 hours, change liquid once; After dialysis finishes, collect dialysis solution, the centrifugal 10min of 10000rpm, gets supernatant, employing CL-4B gel-purified this in conjunction with polysaccharide, and collect conjugate peak; Detect albumen and polyoses content in conjugate solution.

12, streptococcus pneumoniae serotype 19F capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

Take 5.2mg oxidation Pn19F polysaccharide, be dissolved in 1ml pure water, put bar magnet in reaction bulb, on magnetic stirring apparatus, under room temperature, be stirred to polysaccharide and dissolve completely; Add the P2-CRM197A albumen of 3.0mg; After stirring and evenly mixing, take the sodium cyanoborohydride of 4.9mg, add and put in reaction bulb, on magnetic stirring apparatus, keep stirring always; At 18 DEG C of room temperatures, react 24 hours; The sodium borohydride that takes 2.5mg, is added to reaction bulb; At 18 DEG C of calorstats, react 5 hours; Reactant mixture is transferred to bag filter (MWCO12-14000), and at 4 DEG C, to buffer dialysis 5 times, the liquid measure of at every turn dialysing 6L, changes liquid once in every 5 hours; After dialysis finishes, collect dialysis solution, the centrifugal 10min of 10000rpm, gets supernatant, employing CL-4B gel-purified this in conjunction with polysaccharide, and collect conjugate peak; Detect albumen and polyoses content in conjugate solution.

13, streptococcus pneumoniae serotype 23F capsular polysaccharide-P2-CRM197A protein conjugates is synthetic

Take 4.9mg oxidation Pn23F polysaccharide, be dissolved in 1ml pure water, put bar magnet in reaction bulb, on magnetic stirring apparatus, under room temperature, be stirred to polysaccharide and dissolve completely; Add the P2-CRM197A albumen of 5.0mg; The sodium cyanoborohydride that takes 5.1mg, adds and puts in reaction bulb, keeps stirring on magnetic stirring apparatus always; At 18 DEG C of calorstats, react 17 hours; The sodium borohydride that takes 2.5mg, is added to reaction bulb; At 18 DEG C of calorstats, react 5 hours; Reactant mixture is transferred to bag filter (MWCO12-14000), at 4 DEG C, to the dialysis of 0.15M sodium chloride solution, the liquid measure of dialysing 6L at every turn.Within every 5 hours, change liquid once, totally 5 times; After dialysis finishes, collect dialysis solution, the centrifugal 10min of 10000rpm, gets supernatant, employing CL-4B gel-purified this in conjunction with polysaccharide, and collect conjugate peak; Detect albumen and polyoses content in conjugate solution.

Two, 13 kinds of pneumococcal capsular polysaccharide P2CRM197A conjugates of other P2CRM197A protein carrier that contains omnipotent epitope peptide is synthetic

The present invention designs and produces totally 7 kinds of P2CRM197A protein carriers and carrys out synthetic GL-PP conjugate, due to the structural similarity of protein, when synthetic for specific streptococcus pneumoniae serotype polysaccharide, the method adopting is the one in reduction amine method, ADH method or CDAP method.The synthetic method of 13 kinds of pneumococal polysaccharide-P2CRM197A synthetics in prosthomere example is similar, does not enumerate concrete grammar at this.

The 3rd step: preparation and the immunogenic assessment of 13 valency pneumococal polysaccharide P2CRM197A conjugates

Prepare corresponding vaccine by the pneumococal polysaccharide protein conjugates preparing, white mice is carried out inoculation, blood sampling, uses ELISA method to detect polysaccharide antibody concentration and opsono-cytophagic test in serum, assess the immunogenicity of different conjugates.

One, preparation and the immunogenic assessment of 13 valency pneumococal polysaccharide-P2CRM197A conjugates

For the immunogenicity of the synthetic pneumococcal capsular polysaccharide P2CRM197A conjugate of assessment P2CRM197A protein carrier is better than the synthetic pneumococcal capsular polysaccharide CRM197A conjugate of CRM197A protein carrier that does not contain omnipotent epitope peptide, the present invention has synthesized 13 valency pneumococal polysaccharide-CRM197A in contrast, carries out the assessment of immunogenicity reinforced effects.

1, the preparation of 13 valency pneumococal polysaccharide-P2CRM197A combined vaccines and 13 valencys pneumococal polysaccharide-CRM197A combined vaccine

With the Labscale ultrafiltration system of Millipore respectively by 1,3,4,5,6A, 7F, 9V, 14,18C, 19A, it is 40 μ g/ml that 19F and 23F streptococcus pneumoniae serotype capsular polysaccharide protein conjugates solution are concentrated into polysaccharide concentration; 6B serotype conjugate solution concentration is concentrated into polysaccharide concentration and is approximately 80 μ g/ml; Calculate and add the monovalent serum type conjugate solution of respective volume to office preparation bottle according to following form.

In connection with 0.22 μ m film aseptic filtration for thing mixed liquor; Add aseptic aluminum phosphate colloid, final aluminium ion concentration is 125mg/ml; Be settled to final volume with buffer; Fill, 0.5ml/ bottle.

The preparation of the 13 valency pneumococal polysaccharide P2CRM197A combined vaccines that 2, contain other P2CRM197A protein carrier

Joint in reference " preparation and the immunogenic assessments of 13 valency pneumococal polysaccharide-P2CRM197A conjugates " method, the present invention has prepared following 6 kind of 13 valency pneumococal polysaccharide P2CRM197A in conjunction with corresponding vaccine, and in order to immunogenicity assessment experiment, comprise 13Pn-CRM197A-P2,13Pn-P2-CRM197A-P2,13Pn-P2-P2-CRM197A, 13Pn-CRM197A-P2-P2,13Pn-P2-P2-CRM197-P2 and 13Pn-P2-CRM197A-P2-P2.

3, injection white mice inoculation and blood sampling

The CM57 that gets 5-6 week is 70 of mices, every 13 valency pneumococal polysaccharide-P2CRM197A protein binding vaccines prepared by injected in mice, injection capacity be 0.1ml/ prop up mice/time.Inoculation mice is divided into three groups, 13 valency pneumococal polysaccharide-P2CRM197A protein vaccines of one group of injection the present invention preparation, another group inoculation 13 valency pneumococal polysaccharide-CRM197A combined vaccines in contrast, the 3rd group is polysaccharide contrast, and concrete vaccinate and collection immune serum flow sheet are as follows:

Gather mouse blood to centrifuge tube, at room temperature leave standstill 2 hours, under 10000rpm condition centrifugal 10 minutes, draw centrifugal supernatant serum with liquid-transfering gun is careful, be stored in 4 DEG C of refrigerators, to be checked.

4, ELISA method detects polysaccharide antibody concentration in mice serum

Prepare respectively 13 kinds of different serotypes pneumococal polysaccharide storing solution 1mg/ml (in 1 × PBS solution), be stored in 4 DEG C of refrigerators.Dilute serotype pneumococal polysaccharide storage liquid to be checked to coated buffer 2-4 μ g/ml, add the coated solution of 100 μ l and in each hole, be coated with elisa plate, at room temperature overnight incubation.Wash 4 times with washing plate buffer, add 100 μ l sealing buffer, at room temperature hatch 2 hours, wash 4 times with washing plate buffer, can at 4 DEG C, preserve one week.

The corresponding test serum 1:10 of injected in mice combined vaccine and control sample acquisition is diluted to working prototype serum, dilution suitable multiple, joins in elisa plate the first round cumulative volume 200 μ l, start to carry out two times of serial dilutions downwards from first row, at room temperature hatch 2 hours.Wash 4 times with washing plate buffer, add 100 μ l alkali phosphatase enzyme mark sheep anti-mouse antibodies (1:2000 dilution), at room temperature hatch 4 hours.Wash 4 times with washing plate buffer, add 100 μ l phosphoric acid-4-nitro phenyl ester disodium salt substrate solutions, read dish at 405nm.

Specificity streptococcus pneumoniae serotype polysaccharide antibody concentration results in mice serum is as following table:

Result shows, pneumococcal Polysaccharide Conjugate Vaccine of the present invention, and taking P2CRM197A as protein carrier, the immunogenicity of 13 synthetic valency pneumococcal capsular polysaccharide-P2CRM197A conjugates is better than 13 valency pneumococcal capsular polysaccharide CRM197A conjugates.The IgG antibody concentration of injecting anti-specificity pneumococal polysaccharide in the mice serum after three pins is significantly higher than IgG antibody concentration in the serum of injecting a pin and two pins; Inject each serotype antibody concentration after three pins higher than injection the more than 4 times of one pin, meet the standard that WHO improves for GL-PP combined vaccine concentration.With 13 valency pneumococcal capsular polysaccharide-CRM197A conjugate comparisons, the concentration of each serotype antibody of 13 valency pneumococcal capsular polysaccharide-P2CRM197A conjugates, after injection three pins, the polysaccharide specific antibody concentration in mice serum is higher.

5, opsono-cytophagic test (Opsonophagocytic Assay, OPA)

Opsono-cytophagic test is to evaluate 13 valency pneumococal polysaccharide-P2CRM197A combined vaccine fungicidal effectiveness methods, according to UAB-MOPA " the many types of opsonophagocytosis bactericidal assay of streptococcus pneumoniae capsular polysaccharide specific antibody method ", the mouse immune serum obtaining is tested.OPA concentration results is as following table:

Result of the test is visible, inject the merging mouse immune serum of 13 valency pneumococal polysaccharide-P2CRM197A conjugate groups after three pins and the merging mouse immune serum comparison of 13 valencys pneumococal polysaccharide-CRM197A conjugate group, the OPA concentration of its antibody is significantly improved.

Two, the preparation of other 13 valency pneumococal polysaccharide-P2CRM197A conjugate and immunogenic assessment are similar with the method for upper joint " preparation and the immunogenic assessments of 13 valency pneumococal polysaccharide-P2CRM197A conjugates ", the antipolysaccharide antibody IgG concentration results of 13 valency pneumococal polysaccharide-P2CRM197A conjugates that other protein carrier that contains P2 synthesizes is as following table, and this table is just listed the polysaccharide IgG antibody concentration after inoculation three pins.

Result from above table, the immunogenicity of the synthetic 13 valency pneumococal polysaccharide conjugates of the omnipotent epi-position PEPC of all P2 of containing RM197A protein carrier and do not contain the 13 valency pneumococal polysaccharide conjugate comparisons that the omnipotent epi-position PEPC of P2 RM197A protein carrier synthesizes, has obvious enhancing.The immunogenicity of the 13 valency pneumococal polysaccharide P2CRM197 conjugates that the CRM197A protein carrier that contains two omnipotent epitope peptides of P2 synthesizes will be higher than only containing 13 synthetic valency pneumococal polysaccharide CRM197A conjugates of the omnipotent epi-position PEPC of a P2 RM197A protein carrier.When omnipotent P2 in CRM197A protein carrier epitope peptide quantity is increased to three, and the immunogenicity of the synthetic 13 valency pneumococal polysaccharide P2CRM197A conjugates of the protein carrier that contains two omnipotent epitope peptides of P2 is relatively without significant change.Omnipotent P2 in CRM197A protein carrier epitope peptide is added to N-end and there is no difference with the 13 valency pneumococal polysaccharide P2CRM197A conjugate immunogenicities that add to C-end.

List of references

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3、Giannini?G,Rappuoli?R,Ratti?G,The?amino‐acid?sequence?of?two?non‐toxic?mutants?of?diphtheria?toxin:CRM45and?CRM197.Nucleic?acids?research,12:4063‐4069,1984.

Claims

1. can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, it is characterized in that:

2. according to claim 1ly can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, it is characterized in that: in described P2CRM197A, contain X P2,1≤X≤3.

3. according to claim 1ly can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, it is characterized in that: in described P2CRM197A protein carrier, P2 is attached at N-end or the C-end of CRM197A albumen, or is connected to N-end and C-end simultaneously.

4. according to strengthening the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates described in claim 1,2 or 3, it is characterized in that: between described P2 and CRM197A albumen, be to be connected by GSGSG aminoacid sequence.

5. according to claim 1ly can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, it is characterized in that: described gene recombinaton engineering bacteria is the escherichia coli that build by gene recombination method.

6. according to claim 1ly can strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, it is characterized in that: described pneumonia capsular polysaccharide is the capsular saccharides that the streptococcus pneumoniae by cultivating respectively 13 different serotypes obtains.