CN104830908A - Pseudovirus packaging system and application thereof - Google Patents
Pseudovirus packaging system and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering and molecular biology, in particular to a pseudovirus packaging carrier, a pseudovirus packaging system, application of the pseudovirus packaging carrier and the pseudovirus packaging system to preparation of pseudovirus and a pseudovirus preparation method. The pseudovirus packaging system can prepare the pseudovirus efficiently and conveniently, and a powerful tool is provided for research of viruses, preparation of vaccines and the like.
Description
Technical field
The present invention relates to genetically engineered and biology field, be specifically related to pseudovirus package carrier and packaging system, it is for the preparation of the purposes of pseudovirus, and the preparation method of pseudovirus.
Background technology
Pseudovirus (pseudovirus) be viral capsid proteins or envelope protein parcel carry that the heterologous nucleic acids of reporter gene formed be similar to euvirus there is infective virion, because wrapped nucleic acid does not have the ability copying the whole nucleotide sequences forming virus, so pseudovirus only has the infectivity of taking turns, operate safer.
Pseudovirus, mainly through many plasmid co-transfections HEK293T or 293FT cell, copies voluntarily, expresses assembling formation in cell, obtains by collecting the method such as substratum supernatant or lysing cell.
Pseudovirus possesses the infectivity the same with euvirus, and the process entering cell is the same with euvirus, but it does not have the ability copying and produce virus after entering cell, be thus safe from harm, and can more safely detect for neutralizing antibody and antiviral.
Strong to infectivity, pathogenic virus by force, as SARS-CoV, Ebola virus, rabies virus, the research of H7N9 etc. has great danger, and limited amount, and pseudovirus security is good, and can be prepared in a large number by rotaring dyeing technology, for these dangerous high virus researches provide conveniently.
Pseudovirus system has multiple, mainly comprises the pseudovirus built with lentiviral vectors, the pseudovirus formed with viral protein self-assembly, and with the pseudovirus that virogene transformation, insertion reporter gene are formed.Lentiviral vectors grows up based on HIV-1, contain the genetic information required for packaging, transfection, stable integration, can provide all transcribe and pack the whole accessory proteins required for RNA to restructuring pseudovirus, utilize expression vector and packaging plasmid cotransfection cell simultaneously, in cell, carry out virus packaging, packaged virion is secreted in extracellular medium.
The feature of pseudovirus system is the defect overcoming traditional method, makes that detection technique is safer, easy, quick, high-throughput, and the viruses making some itself be difficult to cultivate also can adopt the method for cultivation to detect.
NIH (NIH) provides the plasmid based on two HIV-1, is respectively do not carry the pSG3. Δ env of Fluc gene and carry the NL4-3.Fluc.R-.E-of Fluc gene.Planting brightness brightness, Nie Jianhui etc. utilizes pSG3. Δ env to establish the method for HIV pseudovirus detection neutralizing antibody, lot of domestic and international researchist utilizes NL4-3.Fluc.R-.E-to construct many pseudoviruss in addition, such as SARS-CoV, Ebola virus, rabies virus, influenza virus etc.Because pSG3. Δ env is not containing reporter gene, therefore can only be used for infecting the cells such as the TZM-bl of expressing luciferase, limits the cells infected scope of pseudovirus; And NL4-3.Fluc.R-.E-can expressing luciferase, thus provide the signal of detection, but the promotor that this plasmid is used is HIV long terminal repeat (LTR), expression efficiency is lower.
Summary of the invention
The invention provides a kind of can the pseudovirus package carrier of high expression luciferase and packaging system, specifically comprise the following aspects.
First aspect present invention relates to a kind of pseudovirus package carrier, and it is be operably connected in carrier pSG3. Δ env luciferase gene and the promotor regulating this genetic transcription.
In one embodiment of the invention, it is that the upstream of nef gene in carrier pSG3. Δ env is operably connected luciferase gene and regulate the promotor of this genetic transcription.
In one embodiment of the invention, it is be operably connected between carrier pSG3. Δ env the 11946th is to 14103 luciferase gene and the promotor regulating this genetic transcription.
In a specific embodiments of the present invention, it is be operably connected at carrier pSG3. Δ env the 13967th place luciferase gene and the promotor regulating this genetic transcription.
In one embodiment of the invention, wherein said luciferase is selected from Photinus pyralis LUC, renilla luciferase.
In a specific embodiments of the present invention, the sequence of described firefly luciferase gene is as shown in SEQ ID NO:3.
In one embodiment of the invention, wherein said promotor is CMV promoter, and preferably, the sequence of described CMV promoter is as shown in SEQ ID NO:18.
In one embodiment of the invention, the sequence of the promotor of wherein said luciferase gene and this genetic transcription of adjustment is as shown in SEQ ID NO:6.
In a specific embodiments of the present invention, described pseudovirus package carrier has the sequence as shown in SEQ IDNO:7.
The present invention relates to a kind of pseudovirus packaging system on the other hand, and it comprises pseudovirus package carrier and at least one carrier for expression of eukaryon of any one of first aspect present invention.
The skeleton carrier packed as pseudovirus of the pseudovirus package carrier of wherein said any one of first aspect present invention in embodiments of the invention, the exogenous gene expression carrier that described carrier for expression of eukaryon is packed as pseudovirus.
In one embodiment of the invention, wherein said carrier for expression of eukaryon is that the sequence of CMV promoter in carrier pcDNA3.1 (+) to T7 promotor is replaced with CAG promotor, LTR promotor or include the sequence of human cytomegalic inclusion disease virus main immediate early protein gene (Human cytomegalovirusmajor immediate-early protein gene) enhancers/promoters, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus.
In one embodiment of the invention, the middle CMV promoter of described pcDNA3.1 (+) is the sequence of 229-896 position to the sequence of T7 promotor.
In one embodiment of the invention, the sequence of described CAG promotor is as shown in SEQ ID NO:10.
In one embodiment of the invention, the sequence of described LTR promotor is as shown in SEQ ID NO:13.
In one embodiment of the invention, the wherein said sequence including human cytomegalic inclusion disease virus main immediate early protein gene (Human cytomegalovirus major immediate-early protein gene) enhancers/promoters, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus is as shown in SEQ ID NO:16.
In one embodiment of the invention, described carrier for expression of eukaryon has the sequence as shown in SEQ ID NO:17.
In one embodiment of the invention, wherein said carrier for expression of eukaryon contains foreign gene, and preferably, described foreign gene is the membrane protein gene of virus.
In one embodiment of the invention, described virus is envelope virus, preferably, described envelope virus comprises DNA virus, RNA viruses and retrovirus, such as be selected from coronavirus (such as SARS-CoV), simplexvirus, poxvirus, Hepadna Virus (as hepatitis C virus), inovirus (as Ebola virus), rhabdovirus (such as rabies virus), influenza virus, paramyxovirus is (as Measles virus, respiratory syncytial virus), flavivirus (such as dengue virus), togavirus, bunyavirus, retrovirus.
The invention still further relates to a kind of carrier for expression of eukaryon, it is that the sequence of CMV promoter in carrier pcDNA3.1 (+) to T7 promotor is replaced with CAG promotor, LTR promotor or include the sequence of human cytomegalic inclusion disease virus main immediate early protein gene (Human cytomegalovirus major immediate-earlyprotein gene) enhancers/promoters, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus.
In one embodiment of the invention, the middle CMV promoter of described pcDNA3.1 (+) is the sequence of 229-896 position to the sequence of T7 promotor.
In one embodiment of the invention, the sequence of described CAG promotor is as shown in SEQ ID NO:10.
In one embodiment of the invention, the sequence of described LTR promotor is as shown in SEQ ID NO:13.
In one embodiment of the invention, the wherein said sequence including human cytomegalic inclusion disease virus main immediate early protein gene (Human cytomegalovirus major immediate-early protein gene) enhancers/promoters, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus is as shown in SEQ ID NO:16.
In a specific embodiments of the present invention, described carrier for expression of eukaryon has the sequence as shown in SEQ IDNO:17.
In one embodiment of the invention, it is also containing foreign gene, and preferably, described foreign gene is the membrane protein gene of virus.
In one embodiment of the invention, described virus is envelope virus, preferably, described envelope virus comprises DNA virus, RNA viruses and retrovirus, such as be selected from coronavirus (such as SARS-CoV), simplexvirus, poxvirus, Hepadna Virus (as hepatitis C virus), inovirus (as Ebola virus), rhabdovirus (such as rabies virus), influenza virus, paramyxovirus is (as Measles virus, respiratory syncytial virus), flavivirus (such as dengue virus), togavirus, bunyavirus, retrovirus.
The invention still further relates to host cell, it contains the carrier for expression of eukaryon of the pseudovirus package carrier of any one of first aspect present invention, the pseudovirus packaging system of any one of the present invention or any one of the present invention.
In one embodiment of the invention, described cell is eukaryotic cell, such as, be the cells such as HEK293, HEK293T, HEK293FT.
The invention still further relates to pseudovirus, it is prepared by the host cell of the pseudovirus packaging system of the pseudovirus package carrier of any one of first aspect present invention, any one of the present invention, the carrier for expression of eukaryon of any one of the present invention or any one of the present invention.
The host cell that the invention still further relates to the pseudovirus package carrier of any one of the present invention, the pseudovirus packaging system of any one of the present invention, the carrier for expression of eukaryon of any one of the present invention or any one of the present invention is preparing the purposes in pseudovirus.
In one embodiment of the invention, wherein said pseudovirus is the pseudovirus of envelope virus, described envelope virus comprises DNA virus, RNA viruses and retrovirus, such as be selected from coronavirus (such as SARS-CoV), simplexvirus, poxvirus, Hepadna Virus (as hepatitis C virus), inovirus (as Ebola virus), rhabdovirus (such as rabies virus), influenza virus, paramyxovirus is (as Measles virus, respiratory syncytial virus), flavivirus (such as dengue virus), togavirus, bunyavirus, retrovirus.
The invention still further relates to a kind of preparation method of pseudovirus, it comprises the following steps:
By the carrier for expression of eukaryon cotransfection eukaryotic cell of the pseudovirus package carrier of any one of first aspect present invention and any one of the present invention, collecting cell culture supernatant, namely obtains pseudovirus.
In embodiments of the invention, described eukaryotic cell is such as selected from the cells such as HEK293, HEK293T, HEK293FT.
In one embodiment of the invention, provide a kind of new lentiviral vectors pSG3. Δ env.Fluc carrying Photinus pyralis LUC reporter gene, it is double-stranded cyclic DNA, and length is about 16.4kb.Utilize the HpaI restriction enzyme site of pSG3. Δ env, design In-Fusion connects primer, and pcr amplification luciferase gene, cuts carrier with In-Fusion HD Enzyme Premix by PCR primer and enzyme, connects and obtains pSG3. Δ env.Fluc plasmid.
In one embodiment of the invention, provide a kind of new lentiviral vectors pSG3. Δ env.cmvFluc, it is double-stranded cyclic DNA, and length is about 17.1kb, and it adds CMV promoter to luciferase gene upstream on the basis of gained carrier pSG3. Δ env.Fluc.Particularly, utilize the HpaI restriction enzyme site of pSG3. Δ env, design In-Fusion connects primer, from with pcr amplification the pcDNA3.1 plasmid of luciferase gene with the luciferase gene of CMV promoter, with In-Fusion HD Enzyme Premix, PCR primer and enzyme are cut carrier, connect and obtain pSG3. Δ env.cmvFluc plasmid.
In one embodiment of the invention, provide a kind of new eukaryon expression plasmid pCAG3.1, it is double-stranded cyclic DNA, and length is about 6.5kb.It is on pcDNA3.1+ expression vector basis, replaces CMV promoter by CAG (CMV early enhancer chicken β actin) promotor.According to the sequence of pcDNA3.1+, utilize MluI and NheI restriction enzyme site, design In-Fusion connects primer, and pcr amplification obtains CAG promotor object fragment, with In-Fusion HD EnzymePremix, PCR primer and enzyme are cut carrier, connect and obtain pCAG3.1 plasmid.
In one embodiment of the invention, provide a kind of new eukaryon expression plasmid pLTR3.1, it is double-stranded cyclic DNA, and length is about 5.6kb.Consider that pseudovirus preparation need by eukaryon expression plasmid and HIV skeleton plasmid transfection simultaneously 293T cell, so HIV LTR promotor is substituted into pcDNA3.1+ plasmid, skeleton plasmid can act on LTR at cell inner expression modulin, thus improves the expression amount of LTR downstream gene.According to the sequence of pcDNA3.1+, utilize MluI and NheI restriction enzyme site, design In-Fusion connects primer, and pcr amplification obtains HIV LTR promotor object fragment, with In-Fusion HD Enzyme Premix, PCR primer and enzyme are cut carrier, connect and obtain pLTR3.1 plasmid.
In one embodiment of the invention, provide a kind of new eukaryon expression plasmid pCMV3.1, it is double-stranded cyclic DNA, and length is about 6.3kb.On pcDNA3.1+ expression vector basis, cmv enhancer/promotor, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and human cytomegalic inclusion disease virus main immediate early protein gene intron 1 are replaced original CMV promoter.According to the sequence of pcDNA3.1+, utilize MluI and NheI restriction enzyme site, design In-Fusion connects primer, pcr amplification obtains the object fragment comprising cmv enhancer/promotor, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus, with In-Fusion HD Enzyme Premix, PCR primer and enzyme are cut carrier, connect and obtain pCMV3.1 plasmid.
The beneficial effect of the invention
Pseudovirus packaging system of the present invention efficiently, expediently can prepare pseudovirus, for the research of virus and the preparation etc. of vaccine provide strong instrument.
Accompanying drawing explanation
The structure schematic diagram of Fig. 1 skeleton plasmid pSG3. Δ env.Fluc.
The structure schematic diagram of Fig. 2 skeleton plasmid pSG3. Δ env.cmvFluc.
The structure schematic diagram of Fig. 3 eukaryon expression plasmid pCAG3.1.
The structure schematic diagram of Fig. 4 eukaryon expression plasmid pLTR3.1.
The structure schematic diagram of Fig. 5 eukaryon expression plasmid pCMV3.1.
The comparison of Fig. 6 skeleton plasmid expressing luciferase efficiency.
The comparison of Fig. 7 eucaryon plasmid expressing luciferase efficiency.
The determination of Fig. 8 double-mass model cotransfection ratio.
Pack the effect of pseudovirus between the different eukaryon expression plasmid of Fig. 9 and skeleton plasmid, wherein every picture group is followed successively by pCAG-CVS, pLTR-CVS, pCMV-CVS from left to right.
Pack the effect of pseudovirus between the different eukaryon expression plasmid of Figure 10 and skeleton plasmid, wherein every picture group is followed successively by pCAG-CTN, pLTR-CTN, pCMV-CTN from left to right.
Figure 11 rabies human immunoglobulin is to the inhibition of Kuang Quan street strain pseudovirus.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.The PCR related in embodiment, enzyme are cut, are connected, or competent cell is prepared, transformed equimolecular biological method all with reference to domestic and international pertinent literature.
The structure of embodiment 1 skeleton plasmid pSG3. Δ env.Fluc
Step 1. restriction enzyme HpaI carries out complete degestion, agarose gel electrophoresis to plasmid pSG3. Δ env (NIH is so kind as to give), reclaims test kit reclaim the fragment that enzyme cuts the 14.7kb of rear generation with the glue of Qiagen company.
Step 2. (this laboratory builds, and obtains between BamH1 and XhoI of Fluc gene insertion pcDNA3.1) luciferase gene in the Primestar archaeal dna polymerase pcr amplification pcDNA3.1-Fluc of Takara company, amplimer is:
p1F：
AAGAATAGTGCTGTTGCCACCATGGAAGATGCCAAAAACAT(SEQ IDNO：1)
p1R：
GACATTAAGCAAGTTTTATTACACGGCGATCTTGCCGCCCT(SEQ IDNO：2)
98 DEG C of 1min denaturations, 98 DEG C of 10s, 68 DEG C of 2min extend, totally 35 circulations, and last 72 DEG C extend 10min.Obtain the object fragment of 1.7kb, agarose gel electrophoresis, reclaim test kit with the glue of Qiagen company and reclaim.The sequence of the luciferase object fragment obtained is as shown in SEQ ID NO:3.
gccaccatggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaataa(SEQ ID NO：3)
The luciferase object fragment mixing that the linearized vector that step 1 obtains by step 3. and step 2 obtain, adds In-Fusion HD Enzyme Premix (purchased from Takara), 50 DEG C of reaction 15min in PCR instrument.
Step 4. is by the connection mixed solution of step 3 all direct Transformed E .coil Stbl2 competent cells, and after ice bath 30min, 42 DEG C of heat shock 1min, 30 DEG C of recovery 90min, the LB coated containing 100 μ g/ml ammonia benzyl mycins is dull and stereotyped.Picking list bacterium colony, extracts DNA and enzyme cuts qualification, determines that the luciferase gene of 1.7kb inserts successfully, this plasmid called after pSG3. Δ env.Fluc.The schematic diagram of plasmid construction as shown in Figure 1.
The structure of embodiment 2 skeleton plasmid pSG3. Δ env.cmvFluc
Step 1. restriction enzyme HpaI carries out complete degestion, agarose gel electrophoresis to plasmid pSG3. Δ env, reclaims test kit reclaim the fragment that enzyme cuts the 14.7kb of rear generation with the glue of Qiagen company.
The step 2. Primestar archaeal dna polymerase pcr amplification pcDNA3.1-Fluc middle and upper reaches of Takara company carry the luciferase gene of CMV promoter, and amplimer is:
p3F:AAGAATAGTGCTGTTTGCTTCGCGATGTACGGGCCAGA(SEQ ID NO：4)
p3R:GACATTAAGCAAGTTTTATTACACGGCGATCTTGCCGCCCT(SEQ ID NO：5)
98 DEG C of 1min denaturations, 98 DEG C of 10s, 68 DEG C of 2.5min extend, totally 35 circulations, and last 72 DEG C extend 10min.Obtain the object fragment of 2.3kb, agarose gel electrophoresis, reclaim test kit with the glue of Qiagen company and reclaim.The sequence of carrying the luciferase object fragment of CMV promoter obtained is as shown in SEQ ID NO:6.
tgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggccaccatggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaataa(SEQ ID NO：6)。
Wherein the sequence of CMV promoter is as follows:
Tgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggccacc ( SEQ ID NO：18 )Shown below.
The luciferase object fragment mixing of CMV promoter that what the linearized vector that step 1 obtains by step 3. and step 2 obtained carry, adds In-Fusion HD Enzyme Premix, 50 DEG C of reaction 15min in PCR instrument.
Step 4. is by the connection mixed solution of step 3 all direct Transformed E .coil Stbl2 competent cells, and after ice bath 30min, 42 DEG C of heat shock 1min, 30 DEG C of recovery 90min, the LB coated containing 100 μ g/ml ammonia benzyl mycins is dull and stereotyped.Picking list bacterium colony, extracts DNA and enzyme cuts qualification, determines that the goal gene of 2.3kb inserts successfully, this plasmid called after pSG3. Δ env.cmvFluc.As shown in Figure 2, its complete sequence is as shown in SEQ ID NO:7 for the schematic diagram of plasmid construction.
The structure of embodiment 3 eukaryon expression plasmid pCAG3.1
Step 1. uses restriction enzyme MluI and NheI to plasmid pcDNA3.1+ (purchased from Invitrogen, article No. V790-20, is sequence information see http://www.lifetechnologies.com/order/catalog/product/V79020? ICID=search-product) complete degestion is carried out, agarose gel electrophoresis, reclaims test kit with the glue of Qiagen company and reclaims the fragment that enzyme cuts the 4.7kb of rear generation.
The step 2. Primestar archaeal dna polymerase pcr amplification CAG promoter sequence (Olympic Competition figure in Beijing hundred is so kind as to give) of Takara company, amplimer is:
p5F:GGCCAGATATACGCGCTAGTTATTAATAGTAATCAATTACG(SEQ ID NO：8)
p5R:AAGTTTAAACGCTAGAATTCTTTGCCAAAATGATGAGAC(SEQ ID NO：9)
98 DEG C of 1min denaturations, 98 DEG C of 10s, 68 DEG C of 2min extend, totally 35 circulations, and last 72 DEG C extend 10min.Obtain the object fragment of 1.7kb, agarose gel electrophoresis, reclaim test kit with the glue of Qiagen company and reclaim.The sequence of the CAG promotor obtained is as shown in SEQ ID NO:10.
ctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagggccctttgtgcggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcgtcggtcgggctgcaaccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaatt(SEQ ID NO：10)
The CAG promotor object fragment mixing that the linearized vector that step 1 obtains by step 3. and step 2 obtain, adds In-Fusion HD Enzyme Premix, 50 DEG C of reaction 15min in PCR instrument.
Step 4. is by the connection mixed solution of step 3 all direct Transformed E .coil Stbl2 competent cells, and after ice bath 30min, 42 DEG C of heat shock 1min, 30 DEG C of recovery 90min, the LB coated containing 100 μ g/ml ammonia benzyl mycins is dull and stereotyped.Picking list bacterium colony, extracts DNA and enzyme cuts qualification, determines that the goal gene of 1.7kb inserts successfully, this plasmid called after pCAG3.1.The schematic diagram of plasmid construction as shown in Figure 3.
The structure of embodiment 4 eukaryon expression plasmid pLTR3.1
Step 1. restriction enzyme MluI and NheI carries out complete degestion to plasmid pcDNA3.1+, agarose gel electrophoresis, reclaims test kit reclaim the fragment that enzyme cuts the 4.7kb of rear generation with the glue of Qiagen company.
(NIH be so kind as to give) HIV LTR sequence of step 2. in the Primestar archaeal dna polymerase pcr amplification NL4-3.Fluc.R-.E-plasmid of Takara company, amplimer is:
p7F:GGCCAGATATACGCG GCAGTATCTCGAGACCTAGAA(SEQID NO：11)
p7R:AAGTTTAAACGCTAG TACCTCCTGGGTGCTAGAGA(SEQID NO：12)
98 DEG C of 1min denaturations, 98 DEG C of 10s, 68 DEG C of 1min extend, totally 35 circulations, and last 72 DEG C extend 10min.Obtain the object fragment of 900bp, agarose gel electrophoresis, reclaim test kit with the glue of Qiagen company and reclaim.The LTR sequence obtained is as shown in SEQ ID NO:13.
Gcagtatctcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaacaatgctgcttgtgcctggctagaagcacaagaggaggaagaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattggcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatggaatggatgaccctgagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgacatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatgctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcacccaggaggta(SEQ ID NO：13)
The LTR sequence object fragment mixing that the linearized vector that step 1 obtains by step 3. and step 2 obtain, adds In-Fusion HD Enzyme Premix, 50 DEG C of reaction 15min in PCR instrument.
Step 4. is by the connection mixed solution of step 3 all direct Transformed E .coil Stbl2 competent cells, and after ice bath 30min, 42 DEG C of heat shock 1min, 30 DEG C of recovery 90min, the LB coated containing 100 μ g/ml ammonia benzyl mycins is dull and stereotyped.Picking list bacterium colony, extracts DNA and enzyme cuts qualification, determines that the goal gene of 900bp inserts successfully, this plasmid called after pLTR3.1.The schematic diagram of plasmid construction as shown in Figure 4.
The structure of embodiment 5 eukaryon expression plasmid pCMV3.1
Step 1. restriction enzyme MluI and NheI carries out complete degestion to plasmid pcDNA3.1+, agarose gel electrophoresis, reclaims test kit reclaim the fragment that enzyme cuts the 4.7kb of rear generation with the glue of Qiagen company.
Step 2. uses the Primestar archaeal dna polymerase pcr amplification pDRVISV1.0 carrier of Takara company (see CN 1560259A, Venereal Disease AIDS Preventing Controlling Center China Disease Preventing Con professor Shao Yiming is so kind as to give) in the main immediate early protein gene of human cytomegalic inclusion disease virus (Humancytomegalovirus major immediate-early protein gene) enhancers/promoters, the main immediate early protein gene extron 1 of human cytomegalic inclusion disease virus, human cytomegalic inclusion disease virus main immediate early protein gene intron 1 sequence (CMV IE 5 ' UTR ~ CMV IE intron), amplimer is:
P6F:GGCCAGATATACGCGACCGCCATGTTGACATTGATT(SEQID NO：14)
P6R:AAGTTTAAACGCTAGCGTGTCGACGACGGTGACTGC(SEQID NO：15)
98 DEG C of 1min denaturations, 98 DEG C of 10s, 68 DEG C of 2min extend, totally 35 circulations, and last 72 DEG C extend 10min.Obtain the object fragment of 1.7kb, agarose gel electrophoresis, reclaim test kit with the glue of Qiagen company and reclaim.The CMV promoter sequence obtained is as shown in SEQ ID NO:16.
accgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagactctataggcacacccctttggctcttatgcatgctatactgtttttggcttggggcctatacacccccgcttccttatgctataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactcccctattggtgacgatactttccattactaatccataacatggctctttgccacaactatctctattggctatatgccaatactctgtccttcagagactgacacggactctgtatttttacaggatggggtcccatttattatttacaaattcacatatacaacaacgccgtcccccgtgcccgcagtttttattaaacatagcgtgggatctccacgcgaatctcgggtacgtgttccggacatgggctcttctccggtagcggcggagcttccacatccgagccctggtcccatgcctccagcggctcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggcacagcacaatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtctgaaaatgagcgtggagattgggctcgcacggctgacgcagatggaagacttaaggcagcggcagaagaagatgcaggcagctgagttgttgtattctgataagagtcagaggtaactcccgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacg(SEQ ID NO：16)
Step 1 is obtained the CMV promoter object fragment mixing that linearized vector and step 2 obtain by step 3., adds In-Fusion HD Enzyme Premix, 50 DEG C of reaction 15min in PCR instrument.
Step 4. is by the connection mixed solution of step 3 all direct Transformed E .coil Stbl2 competent cells, and after ice bath 30min, 42 DEG C of heat shock 1min, 30 DEG C of recovery 90min, the LB coated containing 100 μ g/ml ammonia benzyl mycins is dull and stereotyped.Picking list bacterium colony, extracts DNA and enzyme cuts qualification, determines that the goal gene of 1.7kb inserts successfully, this plasmid called after pCMV3.1.As shown in Figure 5, its complete sequence is as shown in SEQ ID NO:17 for the schematic diagram of plasmid construction.
The comparison of embodiment 6 skeleton plasmid expressing luciferase efficiency
According to the specification sheets of lipofectamine2000 transfection reagent, by the skeleton plasmid transfection 293T cell of identical amount, after 48h, cell piping and druming evenly, after dilution, uses Promega ' s Bright-Glo
tMluciferase assay reagents box, detects values of chemiluminescence, the results are shown in Figure 6.Wherein, the luminous result of NL4-3.Fluc.R-.E-, pSG3 Δ env.Fluc is without significant difference, and the illumination effect of pSG3 Δ env.cmvFluc is obviously better than the above two.
The comparison of embodiment 7 eucaryon plasmid expressing luciferase efficiency
For detecting the expression efficiency of pCMV3.1, pCAG3.1, pLTR3.1 eukaryon expression plasmid built, luciferase gene being building up in pCMV3.1, pCAG3.1, pLTR3.1 plasmid respectively, obtaining pCMV-Fluc, pCAG-Fluc, pLTR-Fluc plasmid.According to the specification sheets of lipofectamine2000 transfection reagent, by identical amount plasmid transfection 293T cell, after 48h, cell piping and druming evenly, after dilution, uses Promega ' s Bright-Glo
tMluciferase assay reagents box, detects values of chemiluminescence, the results are shown in Figure 7.Wherein, the fluorescent value of pCMV-Fluc plasmid is the highest, tentatively determines that pCMV3.1 plasmid has higher expression efficiency.
The determination of embodiment 8 double-mass model cotransfection ratio
CVS-11 membranin (GenBank ID:GQ918139) is building up in pcDNA3.1+ plasmid, obtains pcDNA3.1-cvs plasmid.Envelope plasmid pcDNA3.1-cvs and HIV skeleton plasmid (pSG3 Δ env.Fluc) are distinguished transfection 293T cell according to certain mass ratio (2:1,1.5:1,1:1,1:1.5,1:2,1:3), about 48h collects substratum supernatant, the pseudovirus collected is infected 293T cell, after 48h, detects RLU.Result as shown in Figure 8.
As can be seen from the figure, when membranin expression plasmid and skeleton plasmid mass ratio are 1:2, the best pseudovirus that can fall packs effect.
The structure of the mad dog pseudovirus of embodiment 9
Respectively CVS-11 membranin (GenBank ID:GQ918139) and CTN-1 membranin (GenBank ID:FJ959397) are inserted in pCAG3.1, pLTR3.1, pCMV3.1 plasmid respectively, obtain the mad dog membranin such as pCAG-cvs, pLTR-cvs, pCMV-cvs and pCAG-CTN, pLTR-CTN, pCMV-CTN eukaryon expression plasmid.
For detecting the difference of the effect of packing pseudovirus between different eukaryon expression plasmid and skeleton plasmid, respectively two groups of plasmids being carried out combined crosswise cotransfection cultured 293T cell in six porocyte culture plates in advance, after 48h, collecting pseudovirus.Infect 293T cell, after 48h, detect luminous value, obtain the results are shown in Table 1, table 2 and Fig. 9, Figure 10.
The fluorescent value (RLU) of table 1 different plasmid combinations cotransfection cell
| RLU | NL4-3.Fluc.R-.E- | pSG3.Δenv.Fluc | pSG3.Δenv.cmvFluc |
| pCAG-cvs | 39953 | 286220 | 1057333 |
| pLTR-cvs | 144430 | 764614 | 840156 |
| pCMV-cvs | 85698 | 765597 | 1661666 |
The fluorescent value (RLU) of table 2 different plasmid combinations cotransfection cell
| RLU | NL4-3.Fluc.R-.E- | pSG3.Δenv.Fluc | pSG3.Δenv.cmvFluc |
| pCAG-CTN | 73690.5 | 228513.5 | 738745 |
| pLTR-CTN | 53446.5 | 140155 | 654788 |
| pCMV-CTN | 1450.5 | 123711 | 996151 |
Found that: the titre of the pseudovirus that pSG3.cmvFluc obtains as skeleton plasmid is obviously more much higher than other skeleton plasmids, pCMV3.1 expression plasmid and pSG3.cmvFluc skeleton plasmid have best packaging effect, can pack out the mad dog pseudovirus of higher titre.
The pseudovirus preparation of embodiment 10 high titre Kuang Quan street strain and Neutralizing test
By the pCMV-cvs plasmid containing Kuang Quan street strain membranin and pSG3. Δ env.cmvFluc skeleton plasmid 1:2 in mass ratio, utilize lipofectamine2000 transfection reagent cotransfection 293T cell, about 6h changes fresh substratum into, collect substratum supernatant after 48h, packing is frozen for subsequent use in-80 DEG C.Then titration of virus is carried out.Get frozen pseudovirus, 3 times of serial dilutions add (see table 3) in 96 orifice plates, then digest 293T cell, counting 5 × 10
5/ ml, every hole adds 100 μ l, after infecting 48h, detects luminous, utilizes Reed-Muench formulae discovery TCID50 to be 5 × 10
4/ ml.
Table 3
| VC | 3^1 | 3^2 | 3^3 | 3^4 | 3^5 | 3^6 | 3^7 | 3^8 | 3^9 | 3^10 | 3^11 | CC |
| VC | 3^1 | 3^2 | 3^3 | 3^4 | 3^5 | 3^6 | 3^7 | 3^8 | 3^9 | 3^10 | 3^11 | CC |
| VC | 3^1 | 3^2 | 3^3 | 3^4 | 3^5 | 3^6 | 3^7 | 3^8 | 3^9 | 3^10 | 3^11 | CC |
| VC | 3^1 | 3^2 | 3^3 | 3^4 | 3^5 | 3^6 | 3^7 | 3^8 | 3^9 | 3^10 | 3^11 | CC |
| VC | 3^1 | 3^2 | 3^3 | 3^4 | 3^5 | 3^6 | 3^7 | 3^8 | 3^9 | 3^10 | 3^11 | CC |
| VC | 3^1 | 3^2 | 3^3 | 3^4 | 3^5 | 3^6 | 3^7 | 3^8 | 3^9 | 3^10 | 3^11 | CC |
Note: VC is virus control, and CC is cell controls.
Kuang Quan street strain pseudovirus Neutralizing test:
According to titration results, rabies human immunoglobulin (National Institute for Food and Drugs Control's arboviruses Vaccination Room provides rabies human immunoglobulin standard substance and rabies human normal immunoglobulin trial-product) carries out 3 times of serial dilutions respectively, add the pseudovirus of 50TCID50 again, hatch 1h for 37 DEG C, peptic cell, counting 5 × 10
5/ ml, every hole adds 100 μ l, and after infecting 48h, detect luminous, calculate inhibiting rate, result as shown in figure 11.
Utilize graphpad to calculate, the IC50 of immunoglobulin (Ig) is 4 × 10
-4about IU/ml.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (17)
1. a pseudovirus package carrier, it is be operably connected in carrier pSG3. Δ env luciferase gene and the promotor regulating this genetic transcription.
2. the pseudovirus package carrier of claim 1, it is that the upstream of nef gene in carrier pSG3. Δ env is operably connected luciferase gene and regulate the promotor of this genetic transcription.
3. the pseudovirus package carrier of claim 1 or 2, wherein said luciferase is selected from Photinus pyralis LUC, renilla luciferase, and preferably, the sequence of described firefly luciferase gene is as shown in SEQ ID NO:3.
4. the pseudovirus package carrier of claim 1 or 2, wherein said promotor is CMV promoter, and preferably, the sequence of described CMV promoter is as shown in SEQ ID NO:18.
5. the pseudovirus package carrier of any one of claim 1-4, the sequence of the promotor of wherein said luciferase gene and this genetic transcription of adjustment is as shown in SEQ ID NO:6, and preferably, described pseudovirus package carrier has the sequence shown in SEQ ID NO:7.
6. a pseudovirus packaging system, it comprises pseudovirus package carrier and at least one carrier for expression of eukaryon of any one of claim 1-5.
7. the pseudovirus packaging system of claim 6, wherein said carrier for expression of eukaryon is that the sequence of CMV promoter in carrier pcDNA3.1 (+) to T7 promotor is replaced with CAG promotor, LTR promotor or include the sequence of human cytomegalic inclusion disease virus main immediate early protein gene (Human cytomegalovirus major immediate-early protein gene) enhancers/promoters, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus.
8. the pseudovirus packaging system of claim 7, the wherein said sequence including human cytomegalic inclusion disease virus main immediate early protein gene (Human cytomegalovirus majorimmediate-early protein gene) enhancers/promoters, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus is as shown in SEQ ID NO:16, preferably, described carrier for expression of eukaryon has the sequence as shown in SEQID NO:17.
9. the pseudovirus packaging system of any one of claim 6-8, wherein said carrier for expression of eukaryon is also containing foreign gene, and preferably, described foreign gene is the membrane protein gene of virus; Preferably, described virus is envelope virus, further preferably, described envelope virus comprises DNA virus, RNA viruses and retrovirus, such as be selected from coronavirus (such as SARS-CoV), simplexvirus, poxvirus, Hepadna Virus (as hepatitis C virus), inovirus (as Ebola virus), rhabdovirus (such as rabies virus), influenza virus, paramyxovirus (as Measles virus, respiratory syncytial virus), flavivirus (such as dengue virus), togavirus, bunyavirus, retrovirus.
10. carrier for expression of eukaryon, it is that the sequence of CMV promoter in carrier pcDNA3.1 (+) to T7 promotor is replaced with CAG promotor, LTR promotor or include the sequence of human cytomegalic inclusion disease virus main immediate early protein gene (Human cytomegalovirus majorimmediate-early protein gene) enhancers/promoters, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus.
The carrier for expression of eukaryon of 11. claims 10, the wherein said sequence including human cytomegalic inclusion disease virus main immediate early protein gene (Human cytomegalovirus majorimmediate-early protein gene) enhancers/promoters, human cytomegalic inclusion disease virus main immediate early protein gene extron 1 and the main immediate early protein gene intron 1 of human cytomegalic inclusion disease virus is as shown in SEQ ID NO:16, preferably, described carrier for expression of eukaryon has the sequence as shown in SEQ IDNO:17.
The carrier for expression of eukaryon of 12. claims 11, it is also containing foreign gene, and preferably, described foreign gene is the membrane protein gene of virus; Preferably, described virus is envelope virus, further preferably, described envelope virus comprises DNA virus, RNA viruses and retrovirus, such as be selected from coronavirus (such as SARS-CoV), simplexvirus, poxvirus, Hepadna Virus (as, hepatitis C virus), inovirus (as Ebola virus), rhabdovirus (such as rabies virus), influenza virus, paramyxovirus (as Measles virus, respiratory syncytial virus), flavivirus (such as dengue virus), togavirus, bunyavirus, retrovirus.
13. host cells, it contains the carrier for expression of eukaryon of the pseudovirus package carrier of any one of claim 1-5, the pseudovirus packaging system of any one of claim 6-9 or any one of claim 10-12; Preferably, described cell is selected from HEK293, HEK293T, HEK293FT cell.
14. pseudoviruss, it is prepared by the pseudovirus packaging system of the pseudovirus package carrier of any one of claim 1-5, any one of claim 6-9, the carrier for expression of eukaryon of any one of claim 10-12 or the host cell of claim 13.
The pseudovirus package carrier of 15. any one of claim 1-5, the pseudovirus packaging system of any one of claim 6-9, the carrier for expression of eukaryon of any one of claim 10-12 or the host cell of claim 13 are preparing the purposes in pseudovirus.
The purposes of 16. claims 15, wherein said pseudovirus is the pseudovirus of envelope virus, preferably, described envelope virus comprises DNA virus, RNA viruses and retrovirus, such as be selected from coronavirus (such as SARS-CoV), simplexvirus, poxvirus, Hepadna Virus (as hepatitis C virus), inovirus (as Ebola virus), rhabdovirus (such as rabies virus), influenza virus, paramyxovirus is (as Measles virus, respiratory syncytial virus), flavivirus (such as dengue virus), togavirus, bunyavirus, retrovirus.
The preparation method of 17. 1 kinds of pseudoviruss, it comprises the following steps:
By the carrier for expression of eukaryon cotransfection eukaryotic cell of the pseudovirus package carrier of any one of claim 1-5 and any one of claim 10-12, collecting cell culture supernatant, namely obtains pseudovirus.
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