CN1814618A - Japanese schistosome metalloproteuse minic peptide and its screening method and use - Google Patents

Abstract

This invention provides an analog polymorphism amino-acid series of two kinds of Japanese blood fluke metallic protease and its protective immune action to the blood fluke, which utilizes Japanese blood fluke 85kDa metallic protease to sieve phage dodecapeptide library and utilizes phage ELISA, DNA sequencing. Western blot and enzyme activity neutralization test to analyze the selected clons and utilizes an animal experiment to test its immune protection effect, finally, the 10 clons randomly selected are all positive verified by ELISA, five different amino acid serieses are got after test and phage expressing the serieses to clone immune kunming plant mice, in which, two clones can be induced with obvious vermin reduction rate: 31.0% and 31.8% and ovum reduction rate: 52.6% and 54.9% separately, which lays a foundation for researching new Japanese blood fluke vaccines.

Description

Japanese schistosome metalloproteuse minic peptide and screening method thereof and application

Technical field

The present invention relates to two kinds of Japanese schistosome metalloproteuse minic peptides and screening method thereof and the application aspect the anti schistosoma challenge infection, belong to biological technical field.

Background technology

Schistosomicide is that a kind of people beast of serious harm human health suffers from parasitosis altogether, is widely current in the Asia, Africa and Hispanic 74 countries and regions, has 200,000,000 people to be infected approximately, the infected threat of 600,000,000 people.Having five kinds of schistosomicide can infected person and cause the human body schistosomicide, be schistosomiasis japanica at China's popular, this disease be popular in the Yangtze valley and on the south 12 provinces, cities and autonomous regions, bring serious harm for people's healthy and social Economic development.

People are exploring the effective way of control and elimination schistosoma disease always for a long time.Although the multiple prophylactico-therapeutic measures that comprises chemotherapy is arranged at present, but because killing oncomelania and control contagium difficulty are very big, schistosomicide remains a serious public health problem that needs to be resolved hurrily so far, to fundamentally control the popular of schistosomicide, must develop the schistosomicide vaccine, because in the middle of the historical progress of human and disease fight, vaccine is a magic weapon that control disease is propagated, for human beings'health has been made huge contribution always.

Along with the development of biological chemistry and Protocols in Molecular Biology, the increasing effective antigen of schistosomicide is separated, and they can induce certain immune protective efficiency in various animal models.These antigens are mainly albumen and comprise enzyme or glycoprotein, have at present severally to be thought the blood fluke vaccine candidate antigens by WHO.Along with the appearance of gene recombination technology, people recombinate to japonicum gene, express with intestinal bacteria or yeast etc., have developed the japonicum gene engineered vaccine to solve the insufficient problem in source of native protein.Along with immunologic development, immunology theory deepening continuously on molecular level it has been recognized that effective vaccine reaction is by immunogenic antigen decision a small bundle of straw, etc. for silkworms to spin cocoons on is arranged, and just epitope causes.Therefore utilize and have strong immunogenic schistosome antigen epi-position and develop the important channel that blood fluke vaccine has become the blood fluke vaccine development.

Extensively exist various proteolytic enzyme in the schistosomicide body, they played an important role in bilharzial each etap.In these proteolytic enzyme, metalloprotease has special effect, degrades the function that this general proteolytic enzyme had except having catalytic proteins, and it is also to schistosomicide invasion host with produce immune evasion material impact is arranged.Therefore, metalloprotease may be an ideal immunity target.

Proteic epitope can be analyzed and determine to phage random storehouse peptide technology not only, and the mimic epitopes that obtains do not need adjuvant can produce immunne response by inducing mouse yet, therefore, enjoys favor in new generation vaccine research.The present invention utilizes anti-metalloprotease serum screening phage random dodecapeptide storehouse to analyze the binding specificity of anti-metalloprotease serum IgG and metalloprotease epitope, the small peptide of separation simulation metalloprotease epitope, observe these small peptides and in mouse, induce the immune effect of protection against Schistosoma japonicum infection, lay the foundation for developing novel Japanese blood fluke vaccine.

Summary of the invention

The screening method that the object of the present invention is to provide two kinds of Japanese schistosome metalloproteuse minic peptides and a kind of Japanese schistosome metalloproteuse minic peptide is provided.

Another object of the present invention is to provide these two kinds of Japanese schistosome metalloproteuse minic peptides at immanoprotection action to the Schistosoma japonicum challenge infection.

Two kinds of Japanese schistosome metalloproteuse minic peptides of the present invention have following aminoacid sequence respectively

Ser-Asn-Pro-Pro-Gly-Met-Ala-Leu-Ser-Ala-Pro-Pro

And Ile-Thr-Ser-His-Thr-Gly-Yyr-Leu-Gln-Leu-Arg-Leu

Screening method of the present invention is: with SDS-PAGE separating metal proteolytic enzyme from the Schistosoma japonicum vomitus, with the gel immune mouse that contains the 85kDa metalloprotease, after three immunity, measure antibody titers and collect serum, slightly carry IgG with the saturated ammonium sulphate fractionation precipitation, the phage that the specificity bonding force is arranged with screening and IgG is shaken slowly in 37 ℃ in the original peptide storehouse that adds dilution in IgG wraps by plate; Unconjugated phage is removed in washing; Use glycine-hydrochloride buffer (pH2.2) room temperature wash-out again, collect elutriant and measure phage titre, the single phage clone of random choose joins in the ER2738 bacterium liquid, after 37 ℃ of shaking culture with the TBS phage that suspends, with the initiator of this suspension phage as programmed screening, carry out the affine screening of three-wheel altogether, obtain the phage polypeptide strong with the IgG binding specificity, that avidity is high; With in phage E LISA, dna sequencing, Westernblot, the enzymic activity and method such as experiment the clone of three-wheel screening back institute picking is analyzed, and measure its immune protective effect with experimentation on animals.

Japanese schistosome metalloproteuse minic peptide of the present invention can be applicable to schistosomicide challenge infection aspect.

Japanese schistosome metalloproteuse minic peptide provided by the invention has the protective immunity effect to Schistosoma japonicum, promptly is respectively with aminoacid sequence

Ser-Asn-Pro-Pro-Gly-Met-Ala-Leu-Ser-Ala-Pro-Pro

With two kinds of phage clone immunity kunming mices of Ile-Thr-Ser-His-Thr-Gly-Yyr-Leu-Gln-Leu-Arg-Leu, can induce tangible worm reduction rate (being respectively 31.0% and 31.8%) and egg reduction rate (being respectively 52.6% and 54.9%).

By screening to metalloproteuse minic peptide, overcome by natural way and can't obtain or the shortcoming of the metalloprotease epitope that is difficult to obtain, make and obtain effective schistosome antigen in a large number and become possibility, and the polypeptide structure that is obtained is clear, it is clear to form, for the synthetic polypeptide vaccine is laid a good foundation.

Description of drawings

The evaluation figure of the positive phage clone of Fig. 1

Fig. 2 is that the Western bolt of No. 2 and No. 3 phage clones analyzes

Be followed successively by from left to right: M13, No. 2 clone, No. 3 clone, Marker

Fig. 3 is for being the metal proteinase activity electrophoretic analysis figure of substrate with the gelatin

C: normal control group, Incubating Solution are the Tris-HCl damping fluid of 0.1M pH9.0;

2: the gel of hatching with No. 2 phage immune serums;

3: the gel of hatching with No. 3 phage immune serums;

E:EDTA, inhibitors of metalloproteinase, the EDTA of adding 1.0mM in the Incubating Solution;

S: the gel of hatching with the metalloprotease immune serum;

M13: the gel of hatching with the M13 immune serum.

Embodiment

1, main raw and source thereof:

(1) phage random dodecapeptide storehouse (Ph.D-12 ^TMPhage Display Peptide Library contains 1.5 * 10 ¹³Pfu/ml) and host bacterium ER2738 be New England Bioexperiment institute (New England BiolabsInc.) product.

(2) the anti-M13 antibody (HRP-M13Ab) of horseradish peroxidase-labeled is available from Pharmcia company, and the sheep anti-mouse igg of horseradish peroxidase-labeled is a Promega company product.

(3) sodium lauryl sulphate is the import packing of Serva company, methylene diacrylamide is the import packing of Fluka company, it is New England Bioexperiment institute (New England Biolabs Inc.) product that albumen Marker is dyed in the import packing of acrylamide Amresco company, standard molecular weight in advance;

(4) Freund's complete adjuvant is a U.S. Sigma company product; Electrophoresis apparatus is a U.S. Bio-Rad company product, and the E960 microplate reader is an ERMC company product.

(5) laboratory animal and cercaria be used to the escape positive oncomelania of putting cercaria is bought from Jiangsu Province's preventing and treating verminosis institute, and female kunming mice is provided by the laboratory animal department of the Chinese Academy of Sciences of Central South University

(6) preparation of EDTA storage liquid dissolving 2.92 gram EDTA are in 10ml distilled water, and adjust pH to 8.0,1ml divide device-20 ℃.During use, be diluted to working concentration.

(7) table model high speed centrifuge, low-temperature and high-speed whizzer are Hunan Instrument General Factory's product.Other reagent is homemade analytical pure.

2, method and result

(1) the affine screening in thalline peptide storehouse

With SDS-PAGE separating metal proteolytic enzyme from the Schistosoma japonicum vomitus.With the gel immune mouse that contains the 85kDa metalloprotease, measure antibody titers and collect serum through three immunity backs, with the saturated ammonium sulphate fractionation precipitation IgG that slightly purifies, mensuration concentration.Coating buffer (the 0.1MNaHCO that contains 10 μ g IgG with 100 μ l ₃, pH8.5) wrapper sheet is crossed liquid in 4 ℃ of wet boxes, and 5% skimmed milk (be dissolved in TBS[50mMTris, 150mMNaCl, pH7.5]) room temperature sealing 2h, the former peptide storehouse that adds 100 μ l dilution then (contains 10 ¹¹Individual plaque-forming unit) shakes 1h slowly in 37 ℃.With TBS-T (TBS that contains 0.5%V/V Tween-20) washing 8 times, to remove unconjugated phage.Use the every hole of glycine-hydrochloride buffer (pH2.2) the 100 μ l room temperature wash-outs 8 minutes of 0.2M again.Every hole adds 15 μ l neutralizers, and (1MTris-HCl pH9.1) after the neutralization, collects elutriant and measures phage titre.The single phage clone of random choose joined be cultured to OD ₅₉₅In the ER2738 bacterium liquid of ≈ 0.5, behind 37 ℃ of shaking culture 4.5h 10, the centrifugal 15min of 000rpm collects supernatant, and (20%W/V PEG8000 is 2.5MNaCl) more than 4 ℃ of precipitation 2h to add the PEG/NaCl of 1/6 volume.Room temperature 10, the centrifugal 15min of 000rpm precipitation phage removes supernatant, with 1ml TBS suspension phage, put 4 ℃ standby.This suspension phage is carried out the screening of next round as the initiator of programmed screening, carry out the affine screening of three-wheel altogether.After the three-wheel screening, specific phage has obtained nearly 700 times enrichment.The titre of first round elutriant is 2.6 * 10 ³Pfu/ml reaches 1.8 * 10 during to third round ⁶Pfu/ml (table 1)

The concentration effect of each wheel screening pnagus medius of table 1

The screening wheel number	The phage titre (pfu/ml) that adds	The phage titre of wash-out (pfu/ml)
			1 2 3	2×10 11 2×10 11 2×10 11	2.6×10 3 4.5×10 5 1.8×10 6

(2) thalline ELISA identifies positive colony

By ELISA batten (10 μ g/ hole), 4 ℃ are spent the night with metalloprotease immune serum IgG bag, 5% skimmed milk room temperature sealing 2h.Every hole adds 10 ¹²Phage particle, room temperature is placed 2h.Wash the anti-M13 antibody that adds the HRP mark after 3 times with TBS-T, the TMB colour developing, 595nm measures absorbancy.Detected 10 with phage E LISA and paved the single phage clone of random choose the plate and the binding specificity of IgG antibody from the third round elutriant, with original peptide storehouse as negative control (NC).When OD value during more than or equal to the negative control twice, be judged to phage and combine with IgG antibody generation specificity, promptly be accredited as the positive.The result shows that the OD value of the single phage clone of 10 random chooses is compared with negative control, all greater than 2.0, all is positive colony

(Fig. 1)

(3) mensuration of insertion small peptide aminoacid sequence in the thalline

Extract phage single-chain DNA, with-96 ¹¹¹The phage sequencing primer: 5 '- ^HOCCC TCA TAG TTAGCG TAA CG-3 ' checks order through the ABI100 automatic sequencer.Institute's calling sequence Expasy software analysis.Through dna sequence analysis, derive following 5 kinds of different aminoacid sequences to 10 positive phage clones

1.acg tcg ttt ggt agt atg ctt agt aag tgg cag aag

Thr-Ser-Fhe-Gly-Ser-Met-Leu-Ser-Lys-Trp-Gln-Lys

2.agt aat cct ccg ggg atg gct ctt tcg gct ccg cct

Ser-Asn-Pro-Pro-Gly-Met-Ala-Leu-Ser-Ala-Pro-Pro

3.att acg tcg cat acg ggg tat ctg cag ctt cgt ttg

Ile-Thr-Ser-His-Thr-Gly-Tyr-Leu-Gln-Leu-Arg-Leu

4.act ctt gct cat act agt cag att ggg ctt acg gct

Thr-Leu-Ala-His-Thr-Ser-Gln-Ile-Gly-Leu-Thr-Ala

5.atg gag gct tct cat acg cat gcg cgt ccg gcg cct

Met-Glu-Ala-Ser-His-Thr-His-Ala-Arg-Pro-Ala-Pro

(4) immune protective of Schistosoma japonicum challenge infection (animal immune experiment)

70 4 age in week kunming mice be divided into 7 groups at random, wherein inject five kinds of not homotactic phages respectively subcutaneous for 5 groups.Injected dose is 10 ¹²Phage particle divides 3 injections, and control group is injected original peptide storehouse or TBS.Immunity three times.Immunity back two all every mouse infect 40 ± 1 of cercarias through skin of abdomen for the third time.Infect and cutd open adult extremely in back 42 days, calculate worm lotus and every gram liver ovum number (table 2).Statistical software carries out statistical study with the SPSS8.0 edition data.Data are expressed as mean+SD.When P≤0.05, thinking has significance.

The worm reduction rate of each group after the table 2-1 immunity

Group	Number of mice	Obtain borer population	Worm reduction rate	P
					12345 original peptide storehouse TBS	10 10 10 10 10 10 10	23.6±1.6733 18.2±1.9235 18.0±1.4142 25.2±1.7889 24.2±2.2804 24.4±1.8166 26.4±1.9494	10.6 31.0 31.8 4.5 8.3 7.5 -	0.055 0.001 * 0.001 * 0.48 0.073 0.318 -

^*There is significance P value≤0.05

The liver egg reduction rate of each group after the table 2-2 immunity

Group	Number of mice	Obtain borer population	Egg reduction rate	P
					12345 original peptide storehouse TBS	10 10 10 10 10 10 10	99.36±7.28 57.75±3.62 54.94±3.64 106.3±3.84 102.4±2.35 108.8±3.46 121.86±3.47	18.5 52.6 54.9. 12.8 16.0 10.7 -	0.009 * 0.001 * 0.001 * 0.145 0.055 0.200 -

^*There is significance P value≤0.05

Prove that from the animal immunization experiment No. 2 and No. 3 phage clones can be induced and be produced significant worm reduction rate (being respectively 31% and 31.8%) and higher egg reduction rate (reaching 52.6% and 54.9% respectively).

(5) immunoblotting assay

To (contain 10 through the phage clone after the amplification ¹²Phage particle) mix with the SDS sample-loading buffer, put boil 3min in the boiling water after, under non-reduced condition, carry out 10% SDS-PAGE electrophoresis and be transferred on the nitrocellulose membrane.Skim-milk with 5% is membrane closure 2h, PBS-T rinsing 3 times.With the metalloprotease immune serum is one anti-, and the sheep anti-mouse igg of HRP mark is two anti-, the DAB colour developing.The result shows, No. 2 and No. 3 phage clones are through the SDS-PAGE electrophoretic separation and after being transferred to nitrocellulose filter, it contains the gene III albumen that inserts small peptide can band occur at the 67kDa place, and wild-type M13 be unrecognized by schistosoma japonicum infection mice serum (IMS) identification.The result shows, No. 2 and No. 3 clones have good antigenicity (Fig. 2).

(6) with the gelatin be the metal proteinase activity analysis of substrate

Collect the Schistosoma japonicum vomitus, separate the 85kDa metalloprotease.Preparation contains 10% SDS-PAGE separation gel of 0.1% gelatin, and concentrated glue is that the 3.9%. vomitus mixes with the non-reduced sample buffer that does not add DTT afterwards without boiling the sample electrophoresis, and deposition condition is 4 ℃, constant current 18mA.After electrophoresis finished, the TritonX-100 100ml detergent gel with 2.5% 10 minutes was washed 3 times, to remove the SDS in the gel.With the washing of the Tris-HCl damping fluid of 0.1M pH9.0 after 5 minutes, gel vertically is cut into 6 little and carry out mark.Article one, place the Tris-HCl damping fluid overnight incubation of 0.1M pH9.0, overnight incubation in the damping fluid of putting 0.1M pH9.0Tris-HCl+1.0mM EDTA.Other 4 in being put into 0.1M pH9.0Tris-HCl damping fluid before, earlier hatch 2h with No. 2, No. 3, M13 phage immune serum and metalloprotease immune serum (dilution in 1: 50) in 37 ℃ respectively.Coomassie brilliant blue with 0.1% (0.1% Coomassie brilliant blue (the 0.1g Coomassie brilliant blue is dissolved in methyl alcohol: acetic acid: water is in 30: 10: 60 the solution, filters and preserves) dyeing 2h, after the decolouring, observations (Fig. 3)

The result shows, behind No. 2 and No. 3 phage immune mouses, the antibody that is produced is the same with the metalloprotease immune serum have in the effect of 85kDa metal proteinase activity.Among Fig. 32,3 and S (metalloprotease immune serum) bar gel show, the 85kDa metalloprotease respectively in No. 2, No. 3 phage immune serums or metalloprotease immune serum and after, lose the ability of gelatin hydrolysate, thereby the hydrolysis band can not appear, and the M13 immune serum can not in and the 85kDa MMP activities, hydrolysis band (M13 among Fig. 3) appears.The result shows, No. 2 and No. 3 phage small peptides have the immunogen characteristic of 85kDa metalloprotease epi-position.

Sequence table

＜110〉easy Singapore dollar, Ceng Xianfang

＜120〉Japanese schistosome metalloproteuse minic peptide and screening method thereof and application

<160>10

<210>1

<211>36

<212>DNA

＜213〉phage (Phage)

<220>

<400>1

acg tcg ttt ggt agt atg ctt agt aag tgg cag aag 36

Thr Ser Fhe Gly Ser Met Leu Ser Lys Trp Gln Lys

1 5 10

<210>2

<211>36

<212>DNA

＜213〉phage (Phage)

<220>

<400>2

agt aat cct ccg ggg atg gct ctt tcg gct ccg cct 36

Ser Asn Pro Pro Gly Met Ala Leu Ser Ala Pro Pro

1 5 10

<210>3

<211>36

<212>DNA

＜213〉phage (Phage)

<220>

<400>3

att acg tcg cat acg ggg tat ctg cag ctt cgt ttg 36

Ile Thr Ser His Thr Gly Tyr Leu Gln Leu Arg Leu

1 5 10

<210>4

<211>36

<212>DNA

＜213〉phage (Phage)

<220>

<400>4

act ctt gct cat act agt cag att ggg ctt acg gct 36

Thr Leu Ala His Thr Ser Gln Ile Gly Leu Thr Ala

1 5 10

<210>5

<211>36

<212>DNA

＜213〉phage (Phage)

<220>

<400>5

atg gag gct tct cat acg cat gcg cgt ecg gcg cct 36

Met Glu Ala Ser His Thr His Ala Arg Pro Ala Pro

1 5 10

<210>6

<211>12

<212>PRT

＜213〉phage (Phage)

<220>

<400>6

Thr Ser Fhe Gly Ser Met Leu Ser Lys Trp Gln Lys

1 5 10

<210>7

<211>12

<212>PRT

＜213〉phage (Phage)

<220>

<400>7

Ser Asn Pro Pro Gly Met Ala Leu Ser Ala Pro Pro

1 5 10

<210>8

<211>12

<212>PRT

＜213〉phage (Phage)

<220>

<400>8

Ile Thr Ser His Thr Gly Tyr Leu Gln Leu Arg Leu

1 5 10

<210>9

<211>12

<212>PRT

＜213〉phage (Phage)

<220>

<400>9

Thr Leu Ala His Thr Ser Gln Ile Gly Leu Thr Ala

1 5 10

<210>10

<211>12

<212>PRT

＜213〉phage (Phage)

<220>

<400>10

Met Glu Ala Ser His Thr His Ala Arg Pro Ala Pro

1 5 10

Claims (3)

1, two kinds of Japanese schistosome metalloproteuse minic peptides, its amino acid series is respectively

Ser-Asn-Pro-Pro-Gly-Met-Ala-Leu-Ser-Ala-Pro-Pro

And Ile-Thr-Ser-His-Thr-Gly-Yyr-Leu-Gln-Leu-Arg-Leu.

2, the screening method of the described Japanese schistosome metalloproteuse minic peptide of claim 1: with SDS-PAGE separating metal proteolytic enzyme from the Schistosoma japonicum vomitus, with the gel immune mouse that contains the 85kDa metalloprotease, after three immunity, measure antibody titers and collect serum, slightly carry IgG with the saturated ammonium sulphate fractionation precipitation, the original peptide storehouse that in IgG wraps by plate, adds dilution, slowly shake the phage that the specificity bonding force is arranged with screening and IgG in 37 ℃, unconjugated phage is removed in washing, use glycine-hydrochloride buffer room temperature wash-out under pH 2.2 conditions again, collect elutriant and measure phage titre, the single phage clone of random choose joins in the ER2738 bacterium liquid, after 37 ℃ of shaking culture with the TBS phage that suspends, with the initiator of this suspension phage as programmed screening, carry out the affine screening of three-wheel altogether, obtain with the IgG binding specificity strong, the phage polypeptide that avidity is high.With in phage E LISA, dna sequencing, Western blot, the enzymic activity and method such as experiment the clone of three-wheel screening back institute picking is analyzed, and measure its immune protective effect with experimentation on animals.

3, the application of the described Japanese schistosome metalloproteuse minic peptide of claim 1 aspect the schistosomicide challenge infection.

Images