CN101564533A - Hepatitis E protein-nucleic acid composite vaccine and preparation method thereof - Google Patents
Hepatitis E protein-nucleic acid composite vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a hepatitis E protein-nucleic acid composite vaccine, which is characterized in that the vaccine comprises hepatitis E virus recombinant protein and hepatitis E virus recombinant plasmids, wherein the content of the hepatitis E virus recombinant protein is between 5 and 50 mu g/mL, and the content of the hepatitis E virus recombinant plasmids is between 0.1 and 3 mg/mL. The composite vaccine can be effectively used for preventing hepatitis E of people and animals; and animal experiments show that the composite vaccine can effectively induce an organism to synchronously produce humoral immunologic response and cellular immunologic response, two components of the composite vaccine have the effect of mutual promotion, and the composite vaccine is obviously superior to individual protein vaccines or nucleic acid vaccines.
Description
One, technical field
The present invention relates to a kind of protein nucleic acid combination vaccine and preparation method thereof, particularly relate to hepatitis E protein-nucleic acid composite vaccine and preparation method thereof.
Two, background technology
Hepatitis E (Hepatitis E, HE is called for short hepatitis E) is by hepatitis E virus (Hepatitis E Virus, the HEV) viral hepatitis that is caused.HE has become the serious public health problem in the whole world at present, in Southeast Asia, there are 50 fairly large eruption and prevalences of surpassing on ground such as north African and Mexico, and that 11 HE successively took place in China was popular, was listed in because of one of the most serious country of HE morbidity and the dead financial burden that causes.Yet still do not have specificity therapeutic method for HE, also not having active and passive immunity preparation can be for prevention.For the propagation of controlling HE and popular, World Health Organization (WHO) has classified the hepatitis E vaccine as one of vaccine of 21 century override development.
Hepatitis E virus has four kinds of genotype, but has only a serotype, thus successfully produce one safely and effectively the hepatitis E vaccine extensively approved.But owing to there is not sophisticated HEV cell culture system, be difficult to prepare traditional inactivated vaccine and attenuated live vaccine, therefore, can only develop engineered protein vaccine or nucleic acid vaccine.Though still not having commercial hepatitis E vaccine at present comes out, but the research of hev genome engineered vaccine has had very big development, now existing many open reading frame 2 genes or its fragment in different cells successful expression HEV recombiant protein or antigenic peptides, and expression product all has good immunogenicity.The hepatitis E virus dna vaccination is a kind of new generation vaccine, uses when containing the recombiant plasmid immune mouse of HEV genetic fragment, but the cell of inducing specific and humoral immunoresponse(HI).
For infection such as blocking-up hepatitis E virus, hepatitis C virus, HIV (human immunodeficiency virus), plasmodiums, need the dual mediation of cellular immunization and humoral immunization.Yet the vaccine-induced cellular immune level of engineered protein is low, and the immune response strength of nucleic acid vaccine is not high.In order to realize the dual mediation of cellular immunization and humoral immunization, the common research of using of protein vaccine and nucleic acid vaccine has appearred.
Three, summary of the invention
Technical problem: the invention provides a kind of hepatitis E protein-nucleic acid composite vaccine and preparation method thereof, this vaccine is based on hepatitis E virus recombiant protein and hepatitis E recombinant plasmid dna, behind the immune animal, can induce body generation immunne response effectively.Just exempt from DNA booster immunization method with albumen or the independent immunity of nucleic acid and albumen and compare, have the advantage of inducing stronger humoral immunoresponse(HI) and effectively strengthening cellullar immunologic response.
Technical scheme: technical solution of the present invention is: a kind of hepatitis E protein-nucleic acid composite vaccine, it is characterized in that comprising hepatitis E virus recombiant protein and hepatitis E virus recombiant plasmid, wherein the content of hepatitis E virus recombiant protein is 5-50ug/mL, and the content of hepatitis E virus recombiant plasmid is 0.1-3mg/mL.
This vaccine also comprises immunological adjuvant, and this immunological adjuvant is a gel aluminum hydroxide solution, and the concentration range of gel aluminum hydroxide is 0.6-1.5mg/mL.
The amino terminal of HEV recombinant protein and HEV recombinant plasmid all 380 of its open reading frame 2 encoding proteins between 465 amino acids; Its carboxyl terminal all 590 between 650 amino acids, have SEQ ID NO:1 amino acid sequence: RGIALTLFNLADTLLGGLPTELIS SAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDYDN QHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQTTYGSSTNPMYVSDTVTFV NVATGAQGVSRSLDWSKVTLDGRPLMTIQQYSKTFFVLPLRGKLSFWEAGTTKAGY PYNYNTTASDQILIENAAGHRVCISTYTTNLGSGPVSISAVGVLAPHSALAALEDT VDYPARAHTFDDFCPECRALGLQ.
A kind of method that is used to prepare hepatitis E protein-nucleic acid composite vaccine, preparation process is: a. is adsorbed in gel aluminum hydroxide with the hepatitis E virus recombiant protein and placed 18-20 hour for 4 ℃; B. the hepatitis E virus recombiant plasmid is diluted with normal saline; C. again with the solution mixing of a and b gained, adjusting hepatitis E virus recombiant protein quality concentration is 5-50ug/mL, and the mass concentration of hepatitis E virus recombiant plasmid is 0.1-3mg/mL, and the mass concentration of gel aluminum hydroxide is 0.6-1.5mg/mL; The mixed solution of gained is hepatitis E protein-nucleic acid composite vaccine.
Described hepatitis E recombiant protein quality concentration is preferably 10ug/mL;
The mass concentration of described hepatitis E virus recombiant plasmid is preferably 1mg/mL;
The mass concentration of described gel aluminum hydroxide is preferably 1.0mg/mL.
Our defined HE protein-nucleic acid composite vaccine, though contain HE protein vaccine and HE nucleic acid vaccine, and be applied to the prevention of HE, it is different from combined vaccine or polyvalent vaccine.The HE protein-nucleic acid composite vaccine reaches the purpose of prevention hepatitis E by the common immunity of protein-nucleic acid when immunity inoculation.Therefore single with regard to combination vaccine, be meant that then to select the different chemical compositions such as albumen, nucleic acid or polysaccharide of same pathogenic microorganism formulated in proportion, and only at the prevention of the disease that same serotype caused of a kind of disease or same pathogenic microorganism.And combined vaccine is meant the antigenic component that contains two or more Different Kinds of Pathogens biology, is formed by the Producer co-formulated, is used to prevent the vaccine product of two or more various disease.If during also as its indication, then carrier bacterin and coupling vaccine also belong to combined vaccine with the carrier bacterium of carrier bacterin and coupling vaccine or the caused disease of link coupled carrier components.Polyvalent vaccine refers to the vaccine with the enrichment culture thing preparation of some serotype bacterium (poison) strain in a kind of microorganism, only at the different serotypes with a kind of disease.
Beneficial effect: compared with prior art, the present invention has following advantage:
Combination vaccine of the present invention prepares simple and convenient, only hepatitis E virus recombiant protein, hepatitis E virus recombiant plasmid, gel aluminum hydroxide directly need be mixed to shake to shake up in proportion getting final product.
Combination vaccine of the present invention can be used to prevent the hepatitis E of people and animal effectively, zoopery shows, this combination vaccine can effectively induce body to produce humoral immunoresponse(HI) and cellullar immunologic response simultaneously, have mutual promoting action between two kinds of components of combination vaccine, obviously be better than independent protein vaccine or nucleic acid vaccine.
The applied immunization ways of combination vaccine of the present invention is immunity altogether, i.e. both superioritys are separately brought into play in protein vaccine and nucleic acid vaccine immunity jointly, effectively induce body to produce humoral immunoresponse(HI) and cellullar immunologic response simultaneously, have complementary advantages.
The amino terminal of applied hepatitis E virus recombiant protein of combination vaccine of the present invention and hepatitis E virus recombiant plasmid 380 of hepatitis E virus open reading frame 2 coding between 465 amino acids, its carboxyl terminal at 590 between 650 amino acids.
In the hepatitis E protein-nucleic acid composite vaccine of the present invention, the gel aluminum hydroxide of use can buy or adopt known method to be prepared.This adjuvant is well known to those skilled in the art, and is easy to obtain and preparation, and is with low cost.
The preferred embodiment of combination vaccine according to the present invention, combination vaccine comprises hepatitis E virus recombiant protein and hepatitis E virus recombiant plasmid, wherein the content of hepatitis E virus recombiant protein is 5-50ug/mL, the content of hepatitis E virus recombiant plasmid is 0.1-3mg/mL, the concentration range of gel aluminum hydroxide is 0.6-1.5mg/mL, and dosage is 0.1mL.The combination vaccine of preparing can effectively induce body to produce humoral immunoresponse(HI) and cellullar immunologic response simultaneously, has mutual promoting action between two kinds of components of combination vaccine, obviously is better than independent protein vaccine or nucleic acid vaccine.
Four, description of drawings
Fig. 1 is the value of anti-HEV-IgG antibody in unit price matched group and the combination vaccine group mice serum;
Fig. 2 is unit price matched group and combination vaccine group mice serum the 14th all antibody and antibody subclass comparison diagram;
Fig. 3 is that the anti-HEV-IgG antibody of different immunization ways mice serums altogether compares;
Fig. 4 is that different immunization ways mice serum the 14th all anti-HEV-IgG antibody altogether and antibody subclass compare;
Fig. 5 is that hepatitis E protein-nucleic acid composite vaccine group and hepatitis E protein-nucleic acid vaccine replace the anti-HEV-IgG antibody of immune group mice serum relatively;
Fig. 6 is that the combination vaccine group is with alternately immune group mice serum the 16th all anti-HEV-IgG antibody and antibody subclass compare;
Fig. 7 is that the anti-HEV-IgG antibody of various dose group mice serum compares;
Fig. 8 is that various dose group mice serum the 14th all anti-HEV-IgG antibody and antibody subclass compare.
Five, the specific embodiment
Hepatitis E protein-nucleic acid composite vaccine is represented with the HE protein-nucleic acid composite vaccine among the embodiment.
A kind of hepatitis E protein-nucleic acid composite vaccine, comprise hepatitis E virus recombiant protein and hepatitis E virus recombiant plasmid, wherein the content of hepatitis E virus recombiant protein is 5-50ug/mL, and the content of hepatitis E virus recombiant plasmid is 0.1-3mg/mL.
Present embodiment also comprises immunological adjuvant, and this immunological adjuvant is a gel aluminum hydroxide solution, and the concentration range of gel aluminum hydroxide is adjusted into 0.6-1.5mg/mL.
The amino terminal of applied hepatitis E virus recombiant protein of hepatitis E protein-nucleic acid composite vaccine and hepatitis E virus recombiant plasmid 380 of hepatitis E virus open reading frame 2 coding between 465 amino acids, its carboxyl terminal at 590 between 650 amino acids.Its described sequence is: RGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQ DKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTA AEYDQTTYGSSTNPMYVSDTVTFVNVATGAQGVSRSLDWSKVTLDGRPLMTIQQYS KTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVCISTYTTNLG SGPVSISAVGVLAPHSALAALEDTVDYPARAHTFDDFCPECRALGLQ.
A kind of method that is used to prepare above-mentioned hepatitis E protein-nucleic acid composite vaccine:
Before preparing final hepatitis E protein-nucleic acid composite vaccine, the hepatitis E virus recombiant protein is adsorbed in gel aluminum hydroxide, placed 18-20 hour, and mixed with the hepatitis E virus recombiant plasmid again for 4 ℃.
Further illustrate below.
Embodiment 3: hepatitis E virus Recombinant Protein Expression and purification
( 1 ) IV ( GTTATCCAGGACTATGATAATCAACATGAGCAAGACCGCCCTACCCCCTCCCCTGCTCCTTCTCGCCCTTTTTCTGTGCTTCGTGCTAATGATGTGCTTTGGCTCTCTCTCACCGCCGCCGAGTATGATCAGACTACCTACGGCTCTTCTACTAACCCTATGTATGTTTCTGATACTGTAACGTTTGTCAATGTGGCCACTGGCGCCCAGGGGGTTTCGCGCTCTCTGGACTGGTCTAAGGTTACCCTTGATGGGCGTCCACTAACTACTATCCAGCAGTATTCCAAGACTTTCTATGTTCTGCCTCTTCGTGGTAAGCTTTCTTTTTGGGAGGCTGGTACTACTAAAGCCGGCTACCCATACAATTATAATACTACTGCTAGTGATCAGATCCTGATTGAGAATGCAGCTGGCCATCGGGTTTGTATTTCTACTTATACTACTAATTTGGGCTCCGGGCCTGTTTCTATCTCTGCTGTCGGTGTCCTCGCACCCCATTCTGCATTGGCCGTTTTGGAGGACACTGTTGATTACCCT ) ,1 ( 5’-CCC CCC ATG GTT ATC CAG GAC TAT GAT AAT C-3’ ) 2 ( 5’-CCC CTC GAG TCAAGG GTAATC AAC AGT GTC CTC CA-3’ ) ORF2453-631 ( p179 ) 。 Is:94 ℃ of of of of-45 seconds of The PCR condition, 52 ℃ of of of of-45 seconds, 72 ℃ of of of of-50 seconds, 35 circulations.The PCR product reclaims purification through agarose gel, be cloned into plasmid vector pET28 (a)+in.
(2) expression plasmid is transformed among the expression strain E.coli BL-21 (DE3), picking list bacterium colony is with containing the LB culture medium culturing of 50ug kanamycin to OD
550=0.6～0.8, with final concentration is that 0.2～1.0mM IPTG induces, and inducing temperature is 37 ℃, and shaking table shakes speed and is 200rpm, centrifugal collection thalline after 3-4 hour, with cell pyrolysis liquid (50mM Tris-HCl, pH 7.2,300mM NaCl) suspension bacterial sediment, ultrasonication thalline after the freeze thawing 6 times, centrifugal collection supernatant, methods such as reuse ion exchange, glucosan chromatography are carried out purification, use UV at last
280, method such as SDS-PAGE detects.
Embodiment 4: hepatitis E virus construction of recombinant plasmid and detection
With recombiant plasmid pCDNA3.1-179 is template, design Auele Specific Primer 1 (5 '-CTAATGGCTAGCGTTATCCAGGACTATG-3 ', 28bp) and primer 2 (5 '-GGAGGATCCAGGGTAATCAACAGTGT-3 ', 26bp) carry out the p179 encoding gene that pcr amplification obtains to contain NheI and BamHI restriction enzyme site, the PCR reaction condition is as follows: 94 ℃ of pre-degeneration 75s, 94 ℃-50s, 50 ℃-45s, 72 ℃-70s, 35 times circulations, 72 ℃ of extension 8min.1% agarose gel electrophoresis is observed, and the enzyme action product is cut glue purification test kit purification with 3S.
With NheI and BamHI difference double digestion plasmid pVAX and purpose segment p179 encoding gene, behind the 1wt% agarose gel electrophoresis, after the enzyme action product is cut glue purification test kit purification with 3S, carry out coupled reaction with the T4DNA ligase, Transformed E-coil DH5 α competent cell, PCR screening positive clone at first, reuse NheI and BamHI double digestion are identified, the cloning and sequencing of the enzyme action positive.
The plasmid transform bacteria, amplification, the extracting that build carried out then that PCR identifies and indirect immunofluorescence testing goal antigen p179 in external expression.
Embodiment 5: preparation hepatitis E protein-nucleic acid composite vaccine gel aluminum hydroxide adsorbent solution
In order to increase immunogenicity, hepatitis E virus recombiant protein and hepatitis E virus recombiant plasmid are adsorbed in gel aluminum hydroxide: (1) mixes hepatitis E recombiant protein and gel aluminum hydroxide; (2) the hepatitis E virus recombiant plasmid is diluted with normal saline.Solution with (1) and (2) gained mixes again, and adjusting hepatitis E recombiant protein quality concentration is 10ug/mL, and the mass concentration of hepatitis E virus recombiant plasmid is 1mg/mL, and the mass concentration of gel aluminum hydroxide is 1.0mg/mL; The mixed solution of gained is hepatitis E protein-nucleic acid composite vaccine gel aluminum hydroxide adsorbent solution.
Embodiment 6: hepatitis E protein-nucleic acid composite vaccine and the comparative experiments of univalent vaccine immunogenicity
A: laboratory animal grouping and immunization protocol
Select 24 of the female Balb/c mouse inbred liness in age in 6-8 week for use, be divided into 3 groups at random, 8 every group.The A group: hepatitis E protein vaccine group, injection hepatitis E virus recombiant protein (10ug/mL), 0.1mL/ is only; The B group: hepatitis E nucleic acid vaccine group, injection hepatitis E virus recombinant plasmid (1mg/mL), 0.1mL/ are only; The C group: the hepatitis E protein-nucleic acid composite vaccine is immune group altogether, injection hepatitis E virus recombiant protein (10ug/mL)+hepatitis E virus recombinant plasmid (1mg/mL), and 0.1mL/ is only;
B: detection method:
Anti-HEV-IgG antibody and anti-HEV-IgG1, IgG2a antibody in the indirect elisa method detection serum.The concrete operations step is as follows: A. wraps quilt: HEV recombinant antigen MIX166 is diluted the back coated elisa plate with 1mol/L carbamide PBST, and 4 ℃ of refrigerator overnight are placed in 100 μ l/ holes; B. application of sample: the ELISA Plate of having wrapped quilt is with PBST liquid washing 3 times, and button is done, and adds 10 μ l mice serums of 5wt% defatted milk powder PBST 90ul dilution, hatches 40min for 37 ℃; C. add ELIAS secondary antibody: PBST liquid washing 4 times, with sheep anti-mouse igg (volume ratio dilution in 1: 5000) and IgG-1, the IgG-2a (volume ratio dilution in 1: 2500) of the PBST dilution HRP labelling that contains 30wt% glycerol, every hole adds 100 μ l, hatches 40min for 37 ℃; D. colour developing: comprise the blank hole, every hole adds substrate A liquid, each 50 μ l of B liquid, pats mixing, puts 37 ℃ of incubations 10 minutes; E. add stop buffer 50 μ l, pat mixing; F. measure: microplate reader is measured each hole OD450 value with the zeroing of blank hole at wavelength 450nm place, prints measurement result; G. criterion as a result: adopt the ratio representation, measure the ratio of hole OD450 with the OD450 that measures specimen hole and negative specimen, promptly P/N＞2.1 o'clock are judged to the positive, otherwise negative, with OD450 value demonstration antibody horizontal.
C: result
The value of anti-HEV-IgG antibody in table one unit price matched group and the combination vaccine group mice serum (contrast accompanying drawing 1)
| | |
|
|
10W | 12W | 14W | |||
| The A group | 0.124 | 0.338 | 0.424 | 0.563 | 0.807 | 0.947 | 1.048 | ||
| The B group | 0.135 | 0.276 | 0.378 | 0.57 | 0.536 | 0.662 | 0.907 | ||
| The C group | 0.174 | 0.456 | 0.599 | 0.776 | 1.109 | 1.038 | 1.165 |
Table 2 unit price matched group and combination vaccine group mice serum the 14th all antibody and antibody subclass be (contrast accompanying drawing 2) relatively
| IgG | IgG-1 | IgG-2a | IgG-1/IgG-2a | |
| The A group | 1.048 | 0.632 | 0.285 | 2.22 |
| The B group | 0.907 | 0.279 | 0.402 | 0.69 |
| The C group | 1.165 | 0.756 | 0.568 | 1.33 |
As shown in Figure 1, 2: anti-HEV-IgG antibody is higher than the albumen matched group in the protein-nucleic acid composite vaccine group serum, but difference not statistically significant (P＞0.05), and its after immunity for the first time in the serum anti-HEV-IgG antibody just be significantly higher than nucleic acid matched group (P＜0.05) always; We have detected the level of mice serum HEV-IgG antibody subclass IgG1 and IgG2a around the tenth again, the IgG1 level of protein-nucleic acid composite vaccine group and the difference not statistically significant of albumen matched group (P＞0.05), and the level of IgG2a is significantly higher than albumen matched group (P＜0.01), the level of its IgG1 and IgG2a all is significantly higher than nucleic acid matched group (P＜0.01), and the ratio of IgG1 and IgG2a is between between the two.Show that protein-nucleic acid composite vaccine is total to immune mouse and can induces stronger humoral immunoresponse(HI), and significantly strengthen the level of cellullar immunologic response, help the antiviral immunity of body.
Embodiment 7: hepatitis E protein-nucleic acid composite vaccine is immune group and the immune group immunogenicity comparative experiments altogether of hepatitis E protein-nucleic acid univalent vaccine altogether
A: laboratory animal grouping and immunization protocol
Select 16 of the female Balb/c mouse inbred liness in age in 6-8 week for use, be divided into 2 groups at random, 8 every group.The A group: the hepatitis E protein-nucleic acid composite vaccine is immune group altogether, injection hepatitis E virus recombiant protein (10ug/mL)+hepatitis E virus recombinant plasmid (1mg/mL), and 0.1mL/ is only; The B group: hepatitis E protein-nucleic acid univalent vaccine is immune group altogether, and two lower limbs are injected hepatitis E virus recombiant protein (10ug/mL) respectively, and hepatitis E virus recombinant plasmid (1mg/mL), each 0.1mL/ are only.Each organizes laboratory animal respectively at the corresponding vaccine of 0,4,8 all intramuscular injection three times, each 0.1mL.
B: detection method:
Anti-HEV-IgG antibody and anti-HEV-IgG1, IgG2a antibody in the indirect elisa method detection serum.The concrete operations step is as follows: A. wraps quilt: HEV recombinant antigen MIX166 is diluted the back coated elisa plate with 1mol/L carbamide PBST, and 4 ℃ of refrigerator overnight are placed in 100 μ l/ holes; B. application of sample: the ELISA Plate of having wrapped quilt is with PBST liquid washing 3 times, and button is done, and adds 10 μ l mice serums of 5wt% defatted milk powder PBST 90ul dilution, hatches 40min for 37 ℃; C. add ELIAS secondary antibody: PBST liquid washing 4 times, with sheep anti-mouse igg (volume ratio dilution in 1: 5000) and IgG-1, the IgG-2a (volume ratio dilution in 1: 2500) of the PBST dilution HRP labelling that contains 30wt% glycerol, every hole adds 100 μ l, hatches 40min for 37 ℃; D. colour developing: comprise the blank hole, every hole adds substrate A liquid, each 50 μ l of B liquid, pats mixing, puts 37 ℃ of incubations 10 minutes; E. add stop buffer 50 μ l, pat mixing; F. measure: microplate reader is measured each hole OD450 value with the zeroing of blank hole at wavelength 450nm place, prints measurement result; G. criterion as a result: adopt the ratio representation, measure the ratio of hole OD450 with the OD450 that measures specimen hole and negative specimen, promptly P/N＞2.1 o'clock are judged to the positive, otherwise negative, with OD450 value demonstration antibody horizontal.
C: result
The anti-HEV-IgG antibody of the different immunization ways mice serums altogether of table 1 is (contrast accompanying drawing 3) relatively
| | |
|
|
10W | 12W | 14W | |||
| A | 0.174 | 0.456 | 0.599 | 0.776 | 1.109 | 1.038 | 1.165 | ||
| B | 0.174 | 0.668 | 0.774 | 0.853 | 1.119 | 1.059 | 1.23 |
Different immunization ways mice serum the 14th all anti-HEV-IgG antibody altogether of table 2 and antibody subclass be (contrast accompanying drawing 4) relatively
| IgG | IgG-1 | IgG-2a | IgG-1/IgG-2a |
| The A group | 1.165 | 0.756 | 0.568 | 2.28 |
| The B group | 1.23 | 1.032 | 0.414 | 4.322 |
As Fig. 3, shown in 4: protein-nucleic acid composite vaccine is immune group inductive humoral immunoresponse(HI) level of institute and albumen altogether, immune group is close altogether for the nucleic acid univalent vaccine, two groups IgG antibody there was no significant difference (P＞0.05), the protein-nucleic acid composite vaccine IgG1 level of immune group altogether is lower than albumen, the nucleic acid univalent vaccine is immune group altogether, but its difference not statistically significant (P＞0.05), the value of the IgG2a of reacting cells immunne response level then is higher than albumen, the nucleic acid univalent vaccine is immune group (P＜0.01) altogether, the IgG-1/IgG-2a that causes combination vaccine to be total to immune group is starkly lower than univalent vaccine immune group altogether, illustrates that protein-nucleic acid composite vaccine is total to immune group and can induces stronger cellullar immunologic response.
Embodiment 8: hepatitis E protein-nucleic acid composite vaccine group and hepatitis E protein-nucleic acid vaccine replace the comparative experiments of immune group immunogenicity
A: laboratory animal grouping and immunization protocol
Select 40 of the female Balb/c mouse inbred liness in age in 6-8 week for use, be divided into 5 groups at random, 8 every group.The A group: the hepatitis E protein-nucleic acid composite vaccine is immune group altogether, injection hepatitis E virus recombiant protein (10ug/mL)+hepatitis E virus recombinant plasmid (1mg/mL), and 0.1mL/ is only; The B group: hepatitis E protein-nucleic acid vaccine replaces immune group-1 (DDP group), the 0th week and 4 weeks injection hepatitis E virus recombinant plasmid (1mg/mL), and 8 weeks injection hepatitis E virus recombiant protein (10ug/mL), each 0.1mL/ is only; The C group: hepatitis E protein-nucleic acid vaccine replaces immune group-2 (PPD group), the 0th week and 4 weeks injection hepatitis E virus recombiant protein (10ug/mL), and 8 weeks injection hepatitis E virus recombinant plasmid (1mg/mL), each 0.1mL/ is only; The D group: hepatitis E protein-nucleic acid vaccine replaces immune group-3 (DPP group), the 0th week injection hepatitis E virus recombinant plasmid (1mg/mL), and the 4th week and 8 weeks injection hepatitis E virus recombiant protein (10ug/mL), each 0.1mL/ is only; The E group: hepatitis E protein-nucleic acid vaccine replaces immune group-4 (PDD group), the 0th week injection hepatitis E virus recombiant protein (10ug/mL), and the 4th week and 8 weeks injection hepatitis E virus recombinant plasmid (1mg/mL), each 0.1mL/ is only.Each organizes laboratory animal respectively at the corresponding vaccine of 0,4,8 all intramuscular injection three times, each 0.1mL.
B: detection method:
Anti-HEV-IgG antibody and anti-HEV-IgG1, IgG2a antibody in the indirect elisa method detection serum.The concrete operations step is as follows: A. wraps quilt: HEV recombinant antigen MIX166 is diluted the back coated elisa plate with 1mol/L carbamide PBST, and 4 ℃ of refrigerator overnight are placed in 100 μ l/ holes; B. application of sample: the ELISA Plate of having wrapped quilt is with PBST liquid washing 3 times, and button is done, and adds 10 μ l mice serums of 5wt% defatted milk powder PBST 90ul dilution, hatches 40min for 37 ℃; C. add ELIAS secondary antibody: PBST liquid washing 4 times, with sheep anti-mouse igg (volume ratio dilution in 1: 5000) and IgG-1, the IgG-2a (volume ratio dilution in 1: 2500) of the PBST dilution HRP labelling that contains 30wt% glycerol, every hole adds 100 μ l, hatches 40min for 37 ℃; D. colour developing: comprise the blank hole, every hole adds substrate A liquid, each 50 μ l of B liquid, pats mixing, puts 37 ℃ of incubations 10 minutes; E. add stop buffer 50 μ l, pat mixing; F. measure: microplate reader is measured each hole OD450 value with the zeroing of blank hole at wavelength 450nm place, prints measurement result; G. criterion as a result: adopt the ratio representation, measure the ratio of hole OD450 with the OD450 that measures specimen hole and negative specimen, promptly P/N＞2.1 o'clock are judged to the positive, otherwise negative, with OD450 value demonstration antibody horizontal.
C: result
Table 1 hepatitis E protein-nucleic acid composite vaccine group and hepatitis E protein-nucleic acid vaccine replace the anti-HEV-IgG antibody of immune group mice serum relatively (contrast accompanying drawing 5)
| | |
|
|
14W | 16W | 18W | |||
| A | 0.119 | 0.245 | 0.751 | 1.17 | 1.337 | 1.466 | 1.345 | ||
| B | 0.02 | 0.04 | 0.032 | 0.037 | 0.174 | 0.359 | 0.275 | ||
| C | 0.05 | 0.138 | 0.244 | 0.308 | 0.699 | 0.756 | 0.675 | ||
| D | 0.013 | 0.046 | 0.097 | 0.298 | 0.911 | 1.034 | 0.958 | ||
| E | 0.024 | 0.042 | 0.04 | 0.052 | 0.196 | 0.239 | 0.281 |
Table 2 combination vaccine group is with alternately immune group mice serum the 16th all anti-HEV-IgG antibody and antibody subclass relatively (contrast accompanying drawing 6)
| IgG | IgG-1 | IgG-2a | IgG-1/IgG-2a | |
| The A group | 1.466 | 1.195 | 0.608 | 1.965 |
| The B group | 0.359 | 0.144 | 0.233 | 0.618 |
| The C group | 0.756 | 0.507 | 0.203 | 2.496 |
| The D group | 1.034 | 0.651 | 0.444 | 1.466 |
| The E group | 0.239 | 0.084 | 0.102 | 0.824 |
Shown in Fig. 5,6: anti-HEV-IgG antibody is significantly higher than all alternately immune group (P＜0.01) in the protein-nucleic acid composite vaccine group serum; We have detected the level of the 16 all mice serum HEV-IgG antibody subclass IgG1 and IgG2a again, the IgG1 of protein-nucleic acid composite vaccine group and the level of IgG2a are significantly higher than all alternately immune group (P＜0.01) equally, and the ratio of IgG1 and IgG2a is between several persons.Show that protein-nucleic acid composite vaccine is total to immune mouse and can induces the stronger humoral immunoresponse(HI) and the level of cellullar immunologic response simultaneously, but humoral immunoresponse(HI) in the highest flight, and remarkable enhanced cell immunne response helps the antiviral immunity of body.
Embodiment 9: the comparative experiments of hepatitis E protein-nucleic acid composite vaccine various dose group immunogenicity
A: laboratory animal grouping and immunization protocol
Select 24 of the female Balb/c mouse inbred liness in age in 6-8 week for use, be divided into 3 groups at random, 8 every group.The A group: hepatitis E protein-nucleic acid composite vaccine low dose group, injection hepatitis E virus recombiant protein (5ug/mL)+hepatitis E virus recombinant plasmid (0.5mg/mL), 0.1mL/ is only; The B group: dosage group in the hepatitis E protein-nucleic acid composite vaccine, injection hepatitis E virus recombiant protein (10ug/mL)+hepatitis E virus recombinant plasmid (1mg/mL), 0.1mL/ is only; The C group: hepatitis E protein-nucleic acid composite vaccine high dose group, injection hepatitis E virus recombiant protein (20ug/mL)+hepatitis E virus recombinant plasmid (2mg/mL), 0.1mL/ is only.Each organizes laboratory animal respectively at the corresponding vaccine of 0,4,8 all intramuscular injection three times, each 0.1mL.
B: detection method:
Anti-HEV-IgG antibody and anti-HEV-IgG1, IgG2a antibody in the indirect elisa method detection serum.The concrete operations step is as follows: A. wraps quilt: HEV recombinant antigen MIX166 is diluted the back coated elisa plate with 1mol/L carbamide PBST, and 4 ℃ of refrigerator overnight are placed in 100 μ l/ holes; B. application of sample: the ELISA Plate of having wrapped quilt is with PBST liquid washing 3 times, and button is done, and adds 10 μ l mice serums of 5wt% defatted milk powder PBST 90ul dilution, hatches 40min for 37 ℃; C. add ELIAS secondary antibody: PBST liquid washing 4 times, with sheep anti-mouse igg (volume ratio dilution in 1: 5000) and IgG-1, the IgG-2a (volume ratio dilution in 1: 2500) of the PBST dilution HRP labelling that contains 30wt% glycerol, every hole adds 100 μ l, hatches 40min for 37 ℃; D. colour developing: comprise the blank hole, every hole adds substrate A liquid, each 50 μ l of B liquid, pats mixing, puts 37 ℃ of incubations 10 minutes; E. add stop buffer 50 μ l, pat mixing; F. measure: microplate reader is measured each hole OD450 value with the zeroing of blank hole at wavelength 450nm place, prints measurement result; G. criterion as a result: adopt the ratio representation, measure the ratio of hole OD450 with the OD450 that measures specimen hole and negative specimen, promptly P/N＞2.1 o'clock are judged to the positive, otherwise negative, with OD450 value demonstration antibody horizontal.
C: result
The anti-HEV-IgG antibody of table 1 various dose group mice serum is (contrast accompanying drawing 7) relatively
| | |
|
|
10W | 12W | 14W | |||
| The A group | 0.171 | 0.394 | 0.623 | 0.739 | 0.885 | 0.929 | 1.061 | ||
| The B group | 0.174 | 0.456 | 0.599 | 0.776 | 1.109 | 1.038 | 1.165 |
| The C group | 0.109 | 0.378 | 0.641 | 0.843 | 1.224 | 1.274 | 1.473 |
Table 2 various dose group mice serum the 14th all anti-HEV-IgG antibody and antibody subclass be (contrast accompanying drawing 8) relatively
| IgG | IgG-1 | IgG-2a | IgG-1/IgG-2a | |
| The A group | 1.061 | 0.569 | 0.328 | 1.74 |
| The B group | 1.165 | 0.756 | 0.568 | 1.33 |
| The C group | 1.473 | 1.395 | 1.381 | 1.01 |
Shown in Fig. 7,8: three various dose groups of combination vaccine are compared, and along with the increase of immunizing dose, the level of humoral immunoresponse(HI) and cellullar immunologic response all has rising in various degree, and three various dose groups present certain dose-effect relationship.The IgG antibody that low dose group, middle dosage group, high dose group are 3 groups raises successively, IgG1/IgG2a then reduces successively, the IgG of high dose group, IgG2a all are higher than middle dosage group (P＜0.05), the IgG of high dose group, IgG1, IgG2a all are significantly higher than low dose group (P＜0.01), and the IgG of middle dosage group, IgG1, IgG2a are higher than low dose group but difference not statistically significant (P＞0.05).
Sequence table
＜110〉Southeast China University
＜120〉hepatitis E protein-nucleic acid composite vaccine and preparation method thereof
<160>6
<210>1
<211>271
<212>PRT
<213>Hepatitis E Virus
<220>
<221>CDS
<222>(1)...(271)
<400>1
1 RGIALTLFNL ADTLLGGLPT ELISSAGGQL FYSRPVVSAN GEPTVKLYTS VENAQQDKGI
61 AIPHDIDLGE SRVVIQDYDN QHEQDRPTPS PAPSRPFSVL RANDVLWLSL TAAEYDQTTY
121 GSSTNPMYVS DTVTFVNVAT GAQGVSRSLD WSKVTLDGRP LMTIQQYSKT FFVLPLRGKL
181 SFWEAGTTKA GYPYNYNTTA SDQILIENAA GHRVCISTYT TNLGSGPVSI SAVGVLAPHS
241 ALAALEDTVD YPARAHTFDD FCPECRALGL Q
<210>2
<211>537
<212>DNA
<213>Hepatitis E virus
<400>2
1 GTTATCCAGG ACTATGATAA TCAACATGAG CAAGACCGCC CTACCCCCTC CCCTGCTCCT
61 TCTCGCCCTT TTTCTGTGCT TCGTGCTAAT GATGTGCTTT GGCTCTCTCT CACCGCCGCC
121 GAGTATGATC AGACTACCTA CGGCTCTTCT ACTAACCCTA TGTATGTTTC TGATACTGTA
181 ACGTTTGTCA ATGTGGCCAC TGGCGCCCAG GGGGTTTCGC GCTCTCTGGA CTGGTCTAAG
241 GTTACCCTTG ATGGGCGTCC ACTAACTACT ATCCAGCAGT ATTCCAAGAC TTTCTATGTT
301 CTGCCTCTTC GTGGTAAGCT TTCTTTTTGG GAGGCTGGTA CTACTAAAGC CGGCTACCCA
361 TACAATTATA ATACTACTGC TAGTGATCAG ATCCTGATTG AGAATGCAGC TGGCCATCGG
421 GTTTGTATTT CTACTTATAC TACTAATTTG GGCTCCGGGC CTGTTTCTAT CTCTGCTGTC
481 GGTGTCCTCG CACCCCATTC TGCATTGGCC GTTTTGGAGG ACACTGTTGA TTACCCT
<210>3
<211>31
<212>DNA
＜213〉synthetic primer
<400>3
1 CCCCCCATGG TTATCCAGGA CTATGATAAT C
<210>4
<211>35
<212>DNA
＜213〉synthetic primer
<400>4
1 CCCCTCGAGT CAAGGGTAAT CAACAGTGTC CTCCA
<210>5
<211>28
<212>DNA
＜213〉synthetic primer
<400>5
1 CTAATGGCTA GCGTTATCCA GGACTATG
<210>6
<211>26
<212>DNA
＜213〉synthetic primer
<400>6
1 GGAGGATCCA GGGTAATCAA CAGTGT
Claims (7)
1. hepatitis E protein-nucleic acid composite vaccine, it is characterized in that comprising hepatitis E virus recombiant protein and hepatitis E virus recombiant plasmid, wherein the content of hepatitis E virus recombiant protein is 5-50ug/mL, and the content of hepatitis E virus recombiant plasmid is 0.1-3mg/mL.
2. hepatitis E protein-nucleic acid composite vaccine according to claim 1 is characterized in that comprising immunological adjuvant, and this immunological adjuvant is a gel aluminum hydroxide solution, and the concentration range of gel aluminum hydroxide is 0.6-1.5mg/mL.
3. hepatitis E protein-nucleic acid composite vaccine according to claim 1, the amino terminal that it is characterized in that hepatitis E virus recombiant protein and hepatitis E virus recombiant plasmid all 380 of its open reading frame 2 encoding proteins between 465 amino acids, its carboxyl terminal all 590 between 650 amino acids, have SEQ ID NO:1 aminoacid sequence.
4. method that is used to prepare the described hepatitis E protein-nucleic acid composite vaccine of claim 1 is characterized in that preparation process is: a. is adsorbed in gel aluminum hydroxide with the hepatitis E virus recombiant protein and placed 18-20 hour for 4 ℃; B. the hepatitis E virus recombiant plasmid is diluted with normal saline; C. again with the solution mixing of a and b gained, adjusting hepatitis E recombiant protein quality concentration is 5-50ug/mL, and the mass concentration of hepatitis E virus recombiant plasmid is 0.1-3mg/mL, and the mass concentration of gel aluminum hydroxide is 0.6-1.5mg/mL; The mixed solution of gained is hepatitis E protein-nucleic acid composite vaccine.
5. the method for preparing hepatitis E protein-nucleic acid composite vaccine according to claim 4 is characterized in that described hepatitis E recombiant protein quality concentration is 10ug/mL.
6. the method for preparing hepatitis E protein-nucleic acid composite vaccine according to claim 4, the mass concentration that it is characterized in that described hepatitis E virus recombiant plasmid is 1mg/mL.
7. the method for preparing hepatitis E protein-nucleic acid composite vaccine according to claim 4, the mass concentration that it is characterized in that described gel aluminum hydroxide is 1.0mg/mL.
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| CN101085347B (en) * | 2007-06-15 | 2010-05-19 | 东南大学 | Hepatitis A, hepatitis B and hepatitis E combination vaccine |
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