CN1712529A

CN1712529A - Chimeric virus particle vaccine carrier protein, its production and use

Info

Publication number: CN1712529A
Application number: CN 200410049703
Authority: CN
Inventors: 王海平; 王全立; 甘慧; 周勇; 高明; 吕丽萍; 付秋霞; 王怡
Original assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Current assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Priority date: 2004-06-24
Filing date: 2004-06-24
Publication date: 2005-12-28
Anticipated expiration: 2024-06-24
Also published as: CN100537762C

Abstract

Intrinsic virus particle vaccine carrier protein, its production and use are disclosed. The process is carried out by inserting continuous subsequence coding gene fragment containing enzyme cut point into DNA sequence of HBcAg el circular zone, obtaining intrinsic gene, cloning intrinsic gene into expression carrier, expressing, obtaining carrier protein, connecting target antigen epi-position to connecting subsequence zone of carrier protein, and melting to obtain protein. It simplifies design and production of virus particle vaccine.

Description

A kind of mosaic type virus-like particle vaccine carrier albumen and preparation method thereof and application

Technical field

The present invention relates to virus vaccines carrier proteins and preparation method thereof and application, the virus sample particle vaccines that particularly relates to a kind of mosaic type virus-like particle vaccine carrier albumen and preparation method thereof and use this carrier proteins preparation with versatility.

Background technology

Vaccination is the effective means that keeps off infection.According to preparation method's difference, the type of vaccine mainly is divided into inactivated vaccine, attenuated vaccine, recombinant vaccine and nucleic acid vaccine.Comprehensive validity and security comparative analysis at present, recombinant vaccine has more advantage.Because traditional solubility recombinant vaccine that comprises neutralizing epitope can only be induced humoral immunization, therefore limited its range of application.Discovering in recent years, virus-like particle, that is: do not contain viral genome but have the structure similar and immunogenic non-infectious particle to virion, has strong immunogenicity, can induce and produce the comprehensive immune response that comprises body fluid and cell, thereby make vaccine have prevention and therapeutic action concurrently, expanded the notion of traditional vaccine.

Virus sample particle vaccines is one of focus of new generation vaccine development in recent years, and its advantage comprises: (1) immunogenicity is strong; (2) can induce comprehensive immunne response; (3) security is good; (4) stable in properties is difficult for being degraded; (5) be convenient to the identification of immunocyte, immune molecule to epitope; (6) immunity the time need not adjuvant.HBV cAg (HBcAg) contains the main epi-position of organism specific cytotoxic T cell (CTL) and a plurality of helper T cell (Th), B cell advantage epi-position, all can be self-assembled into icosahedral virus-like particles at protokaryon and eukaryotic system, the about 27nm of diameter, the particulate fundamental structural unit is a HBcAg albumen dimer, each HBcAg is made up of 90 or 120 albumen dimers, each monomeric N-terminal 78-83 amino acids zone forms bulge-structure at particle surface, is called e1 ring zone.

At present, use HBcAg and prepare virus sample particle vaccines, the method that is adopted is directly to connect corresponding target epitope in the e1 of HBcAg ring zone, needs many primers of design, just can finish through the multistep polymerization polymerase chain reaction, operation steps is many, and method is loaded down with trivial details and may introduce sudden change.

Summary of the invention

The purpose of this invention is to provide a kind of general, mosaic type virus-like particle vaccine carrier albumen and preparation method thereof efficiently.

Mosaic type virus-like particle vaccine carrier albumen provided by the present invention obtains as follows:

1) the connexon sequence encoding gene fragment that will comprise restriction enzyme site is inserted in the dna sequence dna in HBcAg e1 ring zone, obtains mosaic gene;

2) mosaic gene is cloned in the expression vector, obtains carrier proteins after the expression.

The described connexon sequence of step 1) is normally flexible, and in order not influence the characteristic of contiguous epi-position, the polypeptide of the amino-acid residue that comprises 1-18 GlyPro commonly used is preferably the amino-acid residue that comprises 2-10 GlyPro as connexon.

The insertion of target epitope also includes restriction enzyme site on the connexon for convenience, and restriction enzyme site commonly used is arbitrary two kinds among NgoMIV, XmaI and the PspoMI.

Described HBcAg e1 ring is at the 78-83 of HBcAg N-terminal amino acids place, and insertion Anywhere that can be between these positions is preferably inserted in the zone of 79-83,78-80 or 79-82, more preferably between the 79-80 amino acids.When inserting connexon, can keep the e1 sequence; Perhaps, can lack all or part of of e1 sequence, and replace with the connexon sequence.

Because the HBcAg carboxyl terminal is rich in basic aminoacids, can can disturb the host chromosome metabolism in conjunction with DNA, the security of removing assorted DNA when helping the vaccine purifying and using at human body, used HBcAg will pass through the carboxyl terminal excalation usually, can obtain by the polymerase chain reaction.The carboxyl terminal excalation can be N-terminal the 145th amino-acid residue place to the amino acid moiety of C-end or whole disappearances, the amino acid of disappearance for example can comprise N-terminal the 145th amino-acid residue place to C-end amino acid, N-terminal the 149th amino-acid residue place to the C-end amino acid, N-terminal the 150th amino-acid residue place is to C-end amino acid, N-terminal the 156th amino-acid residue place to the C-end amino acid etc.

Mosaic gene clone's expression vector has multiple choices, as plasmid or virus vector etc., they can comprise a replication origin, one and be used for described mosaic gene expression promoter, a promoter regulation element as enhanser, a transcription termination signal, a translation initiation signal and/or a translation termination signal; Described expression vector also can comprise one or more and plant selectable marker gene, as ampicillin resistance gene or neomycin resistance gene etc.Express used organism and can be intestinal bacteria, yeast or eukaryotic cell etc.

Use preferred version of the present invention, the mosaic gene that obtains has the SEQ ID № in the sequence table: 1 nucleotide sequence; The gained carrier proteins is for having SEQ ID № in the sequence table: 2 amino acid residue sequence.

Second purpose of the present invention provides the virus sample particle vaccines of using gained mosaic type virus-like particle vaccine carrier protein Preparation of the present invention.

Adopt conventional fusion protein technology target epitope to be inserted into the connexon zone of gained carrier proteins, for example the encoding gene with target epitope inserts in the connexon zone corresponding DNA sequence of carrier proteins, through obtaining required virus sample particle vaccines behind protein expression, the purifying by endonuclease reaction equimolecular biology techniques.

The disease that will prevent, treat or diagnose is depended in the selection of target epitope, can be t cell epitope or B cell epitope; The perhaps combination of dissimilar epi-positions is as Th epi-position and B cell epitope or CTL epi-position; The series connection of a plurality of copies of perhaps a kind of epi-position.

Use the present invention and can in carrier proteins, introduce HBVpreS1 or HBVpreS1+preS2 easily, obtain two kinds of Hepatitis B virus vaccine CS1 and CS1S2.

The present invention flexibly connects the e1 ring zone that son inserts HBcAg with one section, obtain a kind of mosaic type virus-like particle vaccine carrier albumen with versatility, when the preparation virus sample particle vaccines, only need corresponding target epitope is inserted into the connexon zone, can obtain required vaccine, the vaccine production method is easy, quick.Obtained vaccine proteantigen epitope display is convenient to the identification of immunocyte and immune molecule in particle surface, can be used as the detection antigen of corresponding antibodies; Under the situation of no adjuvant, can inducement efficient, comprehensively immune response, reach prevention pathogenic infection and treatment infectious diseases (particularly intracellular pathogen infectivity), tumour and autoimmune disorder.The mosaic type virus-like particle vaccine carrier albumen with versatility that utilizes the present invention to make up can be simplified the design and the preparation process of virus sample particle vaccines greatly, is with a wide range of applications.

Description of drawings

Fig. 1 is a mosaic type virus-like particle vaccine carrier albumen clone scheme synoptic diagram

Fig. 2 is a Protein S DS-PSGE electrophorogram

Fig. 3 is the electromicroscopic photograph of PROTEIN C 144Tong

Fig. 4 is the electromicroscopic photograph of PROTEIN C S1

Fig. 5 is the electromicroscopic photograph of PROTEIN C S1S2

Fig. 6 is proteic Western blot photo

Embodiment

The structure of embodiment 1, mosaic type virus-like particle vaccine carrier

1, the amplification mosaic gene makes up the pTC144Tong carrier

Adopt overlapping extension scissors excision (SOE), obtain target gene fragment through the three-wheel pcr amplification.

First round PCR: with the human serum that contains HBV is template, with

P1:5 '-GCGCGGATCCATGGACATTGACCCATATAA-3 ' and

P4:5 '-AAAACTGCAGCTACGGAAGTGTTGATAAGA-3 ' is a primer, carries out pcr amplification, and reaction parameter is: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s then, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate 30,72 ℃ are extended 5min again, are kept at 4 ℃.Adopt agarose gel electrophoresis to reclaim the PCR product, obtain the C144 gene.Be connected with plasmid pGEMT (available from Promega company) after the A tail is gone up in 3 ' end connection of C144 gene according to ordinary method, obtain plasmid pTC144.Adopt conventional CaCl ₂Conversion method imports intestinal bacteria with plasmid pTC144, according to the indigo plant-hickie screening method screening positive clone bacterial strain of routine, adopts plasmid extraction kit (purchase white Promega company) therefrom to extract plasmid pTC144.

Second takes turns PCR: with plasmid pTC144 is template, with

P1:5 '-GCGCGGATCCATGGACATTGACCCATATAA-3 ' and

P2:5 '-CGGACCCGGGCCTGCCGGCGGATCTTCCAAATTA-3 ' is a primer, carries out pcr amplification, reclaims to obtain the PC144-1 gene fragment; With plasmid pTC144 is template, with

P3:5 '-GCAGGCCCGGGTCCGGGCCCGGCATCCAGGGAATTA-3 ' and

P4:5 '-AAAACTGCAGCTACGGAAGTGTTGATAAGA-3 ' is a primer, carries out pcr amplification, reclaims to obtain the PC144-2 gene fragment.

Third round PCR: with PC144-1 and PC144-2 is template, with

P1:5 '-GCGCGGATCCATGGACATTGACCCATATAA-3 ' and

P4:5 '-AAAACTGCAGCTACGGAAGTGTTGATAAGA-3 ' is a primer, carries out pcr amplification, reclaims and obtains mosaic gene C144Tong, and according to the ordinary method cloning and sequencing, sequence is SEQ ID № in the sequence table: 1.According to ordinary method C144Tong is connected on the plasmid pGEMT (available from Promega company), obtains plasmid pTC144Tong.Adopt conventional CaCl ₂Conversion method imports intestinal bacteria with plasmid pTC144Tong, according to the indigo plant one hickie screening method screening positive clone bacterial strain of routine, adopts plasmid extraction kit (available from Promega company) therefrom to extract plasmid pTC144Tong.

2, mosaic type virus-like particle vaccine carrier pQE-C144Tong

With BamHI and PstI double digestion plasmid pTC144Tong, after the recovery C144Tong gene fragment, be connected with the prokaryotic expression carrier pQE30a that cuts processing with same enzyme (available from Qiagen company), obtain plasmid pQE-C144Tong, adopt conventional CaCl then ₂Conversion method transformed into escherichia coli JM109 adopts PCR method screening positive clone, adopts plasmid extraction kit (available from Promega company) therefrom to extract plasmid, and is correct through restriction analysis proof gained plasmid.

The pQE-C144Tong plasmid is adopted conventional CaCl ₂Conversion method transformed into escherichia coli M15 adopts PCR method screening positive clone.Choose the positive colony bacterial strain in two resistance (sulphuric acid kanamycin and the penbritin) substratum of LB by the 1:50 inoculum size, be cultured to OD _600nm=0.4 back adds 1mM inductor IPTG (isopropylthio-), the results bacterium carries out the SDS-PAGE gel electrophoresis after 4 hours, the result as shown in Figure 2,1 road is PROTEIN C 144Tong among the figure, 2 roads are embodiment 2 gained PROTEIN C S1,3 roads are embodiment 3 gained PROTEIN C S1S2, and the M road is middle low molecular weight protein (LMWP) standard.As can be seen from the figure, recombinant protein c 144Tong molecular weight is 16KDa.

Use Ni ²⁺The affinity column of chelating separates, purifying obtains recombinant protein c 144Tong, carries out transmission electron microscope observing after the phospho-wolframic acid negative staining, and photo as shown in Figure 3.As can be seen from the figure, recombinant protein c 144Tong is the particle of big or small homogeneous, the about 27nm of diameter.

Measure recombinant protein c 144Tong aminoacid sequence, the result is SEQ ID № in the sequence table: 2.

Fig. 1 is a mosaic type virus-like particle vaccine carrier albumen clone scheme synoptic diagram, and C144-1 is the 1-79 amino-acid residue fragment of HBcAg N-terminal among the figure; C144-2 is the 80-144 amino-acid residue fragment of HBcAg N-terminal; The connexon sequence fragment of connexon for inserting; P1, P2, P3, P4 are the PCR the primer.

The structure of embodiment 2, hepatitis B virus mosaic type virus-like particle vaccine CS1

With plasmid pGEMT-preS1 (the biotechnology communication, 2004,15 (2): 105) be template, with

P5:5 '-AAAAGCCGGCACCTCTGGGATTCTTTCCCGA-3 ' and

P6:5 '-AAAAGGGCCCGGGTTGAAGTCCCAATCTGGATT-3 ' is a primer, carries out pcr amplification and obtains HBVpreS1 (the gene S1 of 2.1～47AA.) epi-positions.Reclaim gene fragment and, be connected to the versatility mosaic type virus-like particle vaccine carrier pQE-C144Tong that cuts processing with same enzyme, obtain plasmid pQE-CS1 with behind NgoMIV and the PspoMI double digestion.Be transformed into e. coli jm109, adopt PCR method screening positive clone, adopt plasmid extraction kit (available from Promega company) therefrom to extract plasmid, correct through restriction analysis proof gained plasmid.

The pQE-CS1 plasmid is adopted conventional CaCl ₂Conversion method transformed into escherichia coli M15 adopts PCR method screening positive clone.Choose the positive colony bacterial strain in two resistance (sulphuric acid kanamycin and the penbritin) substratum of LB by 1: 50 inoculum size, be cultured to OD _600nm=0.4 back adds 1mM inductor IPTG (isopropylthio-), and the results bacterium carries out SDS-PAGE gel electrophoresis (Fig. 2) after 4 hours, and recombinant protein c S1 molecular weight is 20KDa.

Use Ni ²⁺The affinitive layer purification recombinant protein c S1 of chelating carries out transmission electron microscope observing after the phospho-wolframic acid negative staining, photo as shown in Figure 4.As can be seen from the figure, recombinant protein c S1 is the particle of big or small homogeneous, the about 30nm of diameter.

The structure of embodiment 3, hepatitis B virus mosaic type virus-like particle vaccine CS1S2

With plasmid pGEMT-CS1S2 (institute of Military Medical Science Institute periodical, 2003,27 (1): 40) be template, with

P5:5 '-AAAAGCCGGCACCTCTGGGATTCTTTCCCGA-3 ' and

P7:5 '-AAAAGGGCCCGGGCCACCAGCAGGAAAATATAG-3 ' is a primer, carry out pcr amplification and obtain HBVpreS1 (21～47AA.)+preS2 (gene S1S2 of 133～145AA.) epi-positions, reclaim gene fragment and with behind NgoMIV and the PspoMI double digestion, be connected to same enzyme and cut on the carrier pQEC144Tong of processing, obtain plasmid pQE-CS1S2.Be transformed into e. coli jm109, adopt PCR method screening positive clone, adopt plasmid extraction kit (available from Promega company) therefrom to extract plasmid, correct through restriction analysis proof gained plasmid.

The pQE-CS1S2 plasmid is adopted conventional CaCl ₂Conversion method transformed into escherichia coli M15 adopts PCR method screening positive clone.Choose the positive colony bacterial strain in two resistance (sulphuric acid kanamycin and the penbritin) substratum of LB by 1: 50 inoculum size, be cultured to OD _600nm=0.4 back adds 1mM inductor IPTG (isopropylthio-), and the results bacterium carries out SDS-PAGE gel electrophoresis (Fig. 2) after 4 hours, and gained recombinant protein c S1S2 molecular weight is 23KDa.

Use Ni ²⁺The affinitive layer purification recombinant protein c S1S2 of chelating carries out transmission electron microscope observing after the phospho-wolframic acid negative staining, photo as shown in Figure 5.As can be seen from the figure, recombinant protein c S1S2 is the particle of big or small homogeneous, the about 30nm of diameter.

The antigenicity of embodiment 4, vaccine CS1 and CS1S2 is identified

Immunoblotting (Western-blot) method according to routine detects proteic epitope antigen, the result as shown in Figure 6, the M road is middle low molecular weight protein (LMWP) standard among the figure; 1 road is contrast (empty carrier pQE30a transforms the M15 bacterium); 2 roads are PROTEIN C 144Tong; 3 roads are PROTEIN C S1; 4 roads are PROTEIN C S1S2.The result shows that each epi-position of vaccine all has antigenicity, and is showed in particle surface.

Adopt conventional enzyme linked immunological absorption (ELISA) method to detect proteic epitope antigen, also obtain same conclusion.

The animal immune of embodiment 5, vaccine CS1 and CS1S2

Recombinant protein c 144Tong, CS1 and CS1S2 with physiological saline solution, are mixed with 1 μ g/ μ l concentration, and sterile filtration is standby.Get 24 of female Balb/c mouse, be divided into C144Tong group, CS1 group, CS1S2 group and control group at random, adopt the abdominal injection mode, at every turn to injected in mice C144Tong, CS1, CS1S2 solution 0.1ml, contrast injecting normal saline solution 0.1ml, per two week injections 1 time, inject 3 times after, adopt ordinary method detection mouse antibodies, cytokine and CTL level.The result shows: each epi-position of two kinds of vaccine CS1 and CS1S2 all can be induced production of antibodies, and can induce CTL and induce IFN-γ.

Sequence table

<160>2

<210>1

<211>459

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

atggacattg?acccatataa?agaatttgga?gcttctgtgg?agttactctc?ttttttgcct 60

tctgacttct?ttccttctat?tcgagatctc?ctcgacaccg?cctctgctct?gtatcgggag 120

gccttagagt?ctccggaaca?ttgttcacct?caccatacag?cactcaggca?agctattctg 180

tgttggggtg?agttgatgaa?tctggccacc?tgggtgggaa?gtaatttgga?agatccgccg 240

gcaggcccgg?gtccgggccc?ggcatccagg?gaattagtag?tcagctatgt?caatgttaat 300

atgggcctaa?aaatcagaca?actattgtgg?tttcacattt?cctgtcttac?ttttggaaga 360

gaaactgttc?ttgagtattt?ggtgtctttc?ggagtgtgga?ttcgcactcc?tcccgcttac 420

agaccaccaa?atgcccctat?cttatcaaca?cttccgtag 459

<210>2

<211>152

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>2

Met?Asp?Ile?Asp?Pro?Tyr?Lys?Glu?Phe?Gly?Ala?Ser?Val?Glu?Leu?Leu

1 5 10 15

Ser?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Ile?Arg?Asp?Leu?Leu?Asp

20 25 30

Thr?Ala?Ser?Ala?Leu?Tyr?Arg?Glu?Ala?Leu?Glu?Ser?Pro?Glu?His?Cys

35 40 45

Ser?Pro?His?His?Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu

50 55 60

Leu?Met?Asn?Leu?Ala?Thr?Trp?Val?Gly?Ser?Asn?Leu?Glu?Asp?Pro?Pro

65 70 75 80

Ala?Gly?Pro?Gly?Pro?Gly?Pro?Ala?Ser?Arg?Glu?Leu?Val?Val?Ser?Tyr

85 90 95

Val?Asn?Val?Asn?Met?Gly?Leu?Lys?Ile?Arg?Gln?Leu?Leu?Trp?Phe?His

100 105 110

Ile?Ser?Cys?Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr?Leu?Val

115 120 125

Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr?Arg?Pro?Pro?Asn

130 135 140

Ala?Pro?Ile?Leu?Ser?Thr?Leu?Pro

145 150

Claims

1, a kind of mosaic type virus-like particle vaccine carrier albumen obtains as follows: the connexon sequence encoding gene fragment that 1) will comprise restriction enzyme site is inserted in the dna sequence dna in e1 ring zone of HBcAg, obtains mosaic gene; 2) mosaic gene is cloned in the expression vector, obtains carrier proteins after the expression.

2, carrier proteins according to claim 1 is characterized in that: the described connexon of step 1) is to comprise 1-18 amino-acid residue that repeats GlyPro, is preferably to comprise 2-10 amino-acid residue that repeats GlyPro; Described restriction enzyme site is arbitrary two kinds among NgoMIV, XmaI and the PspoMI; The e1 ring zone of described HBcAg is 79-83 amino-acid residue or the 78-80 amino-acid residue or the 79-82 amino-acid residue of HBcAgN end, is preferably the 79-80 amino-acid residue of HBcAgN end.

3, carrier proteins according to claim 1 and 2 is characterized in that: N-terminal the 145th amino-acid residue place of the described HBcAg of step 1) is to amino acid moiety or whole disappearance of C-end.

4, carrier proteins according to claim 1 and 2 is characterized in that: the described mosaic gene of step 1) has the SEQ ID № in the sequence table: 1 nucleotide sequence; Step 2) described carrier proteins has SEQ ID № in the sequence table: 2 amino acid residue sequence.

5, the proteic preparation method of a kind of mosaic type virus-like particle vaccine carrier comprises the steps: 1) the connexon sequence encoding gene fragment that will comprise restriction enzyme site is inserted in the dna sequence dna in e1 ring zone of HBcAg, obtains mosaic gene; 2) mosaic gene is cloned in the expression vector, obtains carrier proteins after the expression.

6, preparation method according to claim 5 is characterized in that: the described connexon sequence of step 1) is to comprise 1-18 amino-acid residue that repeats GlyPro, is preferably to comprise 2-10 amino-acid residue that repeats GlyPro; Described restriction enzyme site is arbitrary two kinds among NgoMIV, XmaI and the PspoMI; Described HBcAg e1 ring zone is 79-83 amino-acid residue or the 78-80 amino-acid residue or the 79-82 amino-acid residue of HBcAg N-terminal, is preferably the 79-80 amino-acid residue of HBcAg N-terminal.

7, according to claim 5 or 6 described preparation methods, it is characterized in that: N-terminal the 145th amino-acid residue place of the described HBcAg of step 1) is to amino acid moiety or whole disappearance of C-end.

8, according to claim 5 or 6 described preparation methods, it is characterized in that: the described mosaic gene of step 1) has the SEQ ID № in the sequence table: 1 nucleotide sequence; Step 2) described carrier proteins has SEQ ID № in the sequence table: 2 amino acid residue sequence.

9, application rights requires the virus sample particle vaccines of 4 described mosaic type virus-like particle vaccine carrier protein Preparation, is target epitope and described carrier proteins are merged the protein that forms.

10, virus sample particle vaccines according to claim 9 is characterized in that: described target epitope is HBVpreS1 or HBVpreS1+preS2.