CN101579527B

CN101579527B - HLA Specific human cytomegalovirus Multi-epitope adenovirus DNA Vaccine of Chinese population

Info

Publication number: CN101579527B
Application number: CN 200910016073
Authority: CN
Inventors: 马道新; 纪春岩; 赵平; 薛付忠
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2009-06-24
Filing date: 2009-06-24
Publication date: 2010-10-27
Anticipated expiration: 2029-06-24
Also published as: CN101579527A

Abstract

The present invention discloses an HLA Specific human cytomegalovirus Multi-epitope adenovirus DNA Vaccine of Chinese population, which consists of human cytomegalovirus tandem sequence CTL*Th and adenovirus expression vector pAd5F35, wherein the human cytomegalovirus tandem sequence CTL*Th comprises a nucleotide sequence represented by SEQ ID No.1. The nucleotide sequence comprises the nucleotidesequences of 83 antigen epitopes of 15 encoding proteins of human cytomegalovirus. Furthermore, the sequence covers 14 HLA I allelomorphic gene sites and 7 HLA II allelomorphic gene sites. The coverage percent of the HCMV tandem epitope DNA vaccine representing excellent HLA is 92.07 percent in average, and is used for preventing the forming of HCMV disease in the steps of adhering, duplicating and re-activating of the virus. The in vetro experiment shows that the vaccine of the invention has excellent immune effect and overcomes the defects of inferior immunogenicity and limited immune rangeof the prior HCMV vaccine.

Description

The special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA

Technical field

The present invention relates to nucleic acid vaccine and structure thereof and application, particularly a kind of Human Cytomegloviru (HCMV) nucleic acid vaccine is the special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA and structure and application.

Background technology

The HCMV disease has become organ transplantation and the common and important complication of intrauterine infection, is to cause graft failure and foetal death and odd-shaped major reason.There are shortcomings such as weak, the immune scope of immunogenicity is limited to, immunoreation is little in existing HCMV vaccine, can not prevent and treat the HCMV viral infection very effectively, mainly be because: the part that really can cause immunne response in (1) antigen molecule is and the bonded epitope of host human leukocyte antigen (HLA) molecular specificity, is the most basic essential 26S Proteasome Structure and Function unit that induces immune response; And HCMV genome total length 235kb, 165 antigen genes of encoding, epitope are extremely complicated, the immune response that the contained limitation epitope of single antigen molecule causes a little less than, can't prevent or eliminate HCMV fully infects; (2) different HCMV epitopes offer activate immunity to reply by different HLA gene locis, however HLA genetic background complexity among the crowd, and single antigen molecule is difficult to cover the different whole crowd in HLA site.Though there is the scholar to consider to use the former vaccine of multi-resistance, the immunopathogenesis reaction that the limited capacity of carrier and high molecular weight protein may cause has also limited the application of a plurality of antigens in identical carrier.How to strengthen the immunogenicity of nucleic acid vaccine greatly under limited carrier capacity, and the nucleic acid vaccine immunity of preparation simultaneously replying crowd's broad covered area, is the difficult point of current HCMV nucleic acid vaccine research.Abroad to the research of HCMV epiposition vaccine at immunity covering crowd be European and African crowd; their HLA genetic background is different with Chinese population HLA distribution situation; the epiposition vaccine of its development is difficult to be offered by the HLA molecule at Chinese population, can not cause effective protective immune response.The present domestic report of not seeing relevant HCMV polyepitope vaccines as yet, and the spatial distribution of Chinese population and nationality is extremely inhomogeneous, the spatial distribution of crowd HLA gene frequency and haplotype frequency presents highly heterogeneous, thereby development is the key point of China HCMV prevention and control field at the powerful HCMV series connection epiposition vaccine of the immune effect of Chinese population HLA characteristic distributions.

Another key factor of vaccine research is the selection of vaccine carrier, over nearly 20 years, have only a few virus successfully to be transformed into gene transfer vector and to have carried out application in various degree as retrovirus (comprising HIV virus), adenovirus, adeno-associated virus, herpesvirus (comprising herpes simplex virus, vaccinia virus and Epstein-Barr virus).The viral vector that is used for gene therapy and vaccine should possess following primary condition: 1. carry exogenous gene and can be packaged into virion; 2. the transfer of mediate foreign gene and expression; 3. not pathogenic to human body; 4. in environment, can not cause propagation and propagation.Wherein replication-defective adenoviral expression vector pAd5F35 development under Japanese Hiroyuki Mizuguchi professor's research is rapid, be widely used in the research of viral vaccine, tumor vaccine, pAd5F35 can be applied in the clinical research by drugs approved by FDA.The gene transfer of adenovirus Ad5F35 mediation has the following advantages: 1. can express destination protein in people's cell quick, high-levelly, because containing the recombinant dna of genes of interest is free on outside the cellular genome, unconformity is gone into host genome, so destination protein matter obtains instantaneous high level expression, studies show that adenovirus Ad5F35 is better than Ad5 type adenovirus at the transduction efficiency of people's hemopoietic progenitor cell, dendritic cell, mescenchymal stem cell, similar with Ad35 type adenovirus, and have hypotoxicity; 2. adenovirus Ad5F35 transduces into cell via cell surface receptor CD46, and the CD46 receptor all has expression on nucleated cell; 3. exogenous gene can be forwarded in division and the nondividing cell and express; 4. can insert external source fragment up to 8kb.

Summary of the invention

The purpose of this invention is to provide the special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of a kind of Chinese population HLA and structure and application.

For reaching above purpose, the present invention is according to HLA I accumulation phenotypic frequency spatial prediction system's (" 2005 the 21st the 2nd phases of volume of IMMUNOLOGY KEY WORDS INDEX of Chinese polyepitope vaccines design, P136), selected 14 HLA gene locis combination is predicted that the coverage rate that is presented at Chinese population reaches 92.07%.

The applicant has selected 15 kinds of antigen proteins of HCMV, 83 epi-positions in these antigens, have been chosen, wherein at 76 of the CTL epi-positions of above-mentioned 14 HLA I gene locis, at 7 of t helper cell (Th) epi-positions of HLA II gene loci, by starting cell and humoral immunoresponse(HI) at above-mentioned epitope, confirmation in the sticking of virus, duplicate, assembling, immunologic escape and again in each process such as activation, can prevent the formation of HCMV disease.

The special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA of the present invention, form by human cytomegalic inclusion disease virus nucleotide tandem sequence CTLTh and adenovirus expression carrier pAd5F35, it is characterized in that, described human cytomegalic inclusion disease virus nucleotide tandem sequence CTLTh contains the nucleotide sequence shown in the SEQ ID No.1, this nucleotide sequence comprises the nucleotide sequence of 83 epitopes of 15 kinds of encoding proteins of human cytomegalic inclusion disease virus, and this sequence covers 14 HLA I allele sites and 7 HLA II allele sites.

Wherein, 15 kinds of encoding proteins of above-mentioned human cytomegalic inclusion disease virus are meant pp28, pp50, pp65, pp150, pp71, gH, gB, IE-1, IE-2, US2, US3, US6, US11, UL16 and UL18; Described 14 HLA I allele sites are meant A2, A24, A1, A3, A11, A68, B44, B7, A23, A26, B35, B38, B8 and B27; Described 7 HLAII allele sites are meant HLA-DR1,3,4,7,11,13 and 15.

As table 1, shown in 2, the epitope sequences of its coding is respectively from 15 kinds of antigen protein (pp28 of human cytomegalic inclusion disease virus, pp50, pp65, pp150, pp71, gH, gB, IE-1, IE-2, US2, US3, US6, US11, UL16 and UL18), respectively in the sticking of virus, duplicate, assembling, immunologic escape and express in the activation process again; The SYFPEITHI software prediction shows, they can with 14 HLA I allele sites (A2, A24, A1, A3, A11, A68, B44, B7, A23, A26, B35, B38, B8 and B27) and 7 HLA II allele sites (HLA-DR1,3,4,7,11,13,15) stable bond and being limited, the Chinese population coverage rate is up to 92.07% (75.49-99.99%).Use MAPPP proteolytic cleavage prognoses system and recombiant protein is predicted the result shows that the polypeptide of generation can be hydrolyzed into the epi-position of expection.

The preparation method of the special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA of the present invention, its epi-position prediction, gene recombinaton, viral packaging step are as follows:

(1), utilize Chinese HLA I to accumulate phenotypic frequency spatial prediction system, to selected HCMV epi-position bonded 14 HLA gene locis (A2, A24, A1, A3, A11, A68, B44, B7, A23, A26, B35, B38, B8 and B27) make up and predict, show the coverage rate average out to 92.07% (Fig. 1) of the special HCMV series connection epi-position nucleic acid vaccine of HLA at Chinese population.15 kinds of antigen proteins (pp28, pp50, pp65 at HCMV, pp150, pp71, gH, gB, IE-1, IE-2, US2, US3, US6, US11, UL16 and UL18) in choose 83 epi-positions, wherein at 76 of the CTL epi-positions (table 1) of above-mentioned 14 HLA I gene locis, at 7 of the Th epi-positions (table 2) of HLA II gene loci, in theory can be in the formation of duplicating, prevent the HCMV disease of each stage control virus.Use DNAMAN software, Kozak rule and PubMedNucleotide NC_001347.Human herpesvirus 5 strain AD169 HCMV genomes and drawn the special HCMV series connection epi-position nucleotide sequence of covering Chinese population HLA, added the Kozak sequence in origin or beginning, can obtain the multi-epitope nucleotides sequence and be listed in efficiently expressing in the carrier for expression of eukaryon; End has added 6 His sequence labels, can use the expression of Western blot and cellular immunization chemical method detection HCMV polyepitope vaccines by 6 * His antibody; Two ends have added Nhe I and Not I restriction enzyme site, can insert the multiple clone site of shuttle vector pHMCMV5 carrier, help preparing the special HCMV series connection epi-position adenovirus vector nucleic acid vaccine of Chinese population HLA.

(2), adopt gene splicing by overlap extension (SOEing) and round pcr to successfully synthesize HCMV polyepitope vaccines full-length gene CTLTh (its nucleotide sequence is shown in SEQ ID No.1).Utilize homologous recombination technique, the CTLTh gene is inserted the Nhe I/Not I restriction enzyme site of shuttle vector pHMCMV5, make up shuttle vector pHMCMV5-CTLTh, use Nhe I/Not I restriction analysis and sequencing analysis and identify the purpose carrier, confirm successfully to have made up shuttle vector pHMCMV5-CTLTh.Utilize I-CeuI/PI-SceI double digestion and extracorporeal recombination that the expression cassette that comprises CMV promoter, genes of interest and SV40 polyA tail on the shuttle vector is inserted adenovirus expression carrier pAd5F35, make up recombinant adenovirus vaccine expression vector Ad5F35-CTLTh, use Xho I and I-Ceu I/PI-SceI restriction analysis and sequencing analysis and identify the purpose carrier, confirm successfully to have made up recombinant adenovirus vaccine expression vector pAd5F35-CTLTh.

(3), use PacI linearisation recombinant adenoviral expressing vector pAd5F35-CTLTh, with liposome 2000 transfection HEK293, be packaged into recombinant adenovirus Ad5F35-CTLTh, wait to cultivate and collected virus when the CPE effect occurring in 10 ± 1 days; Use the HEK293 cell recombinant adenovirus Ad5F35-CTLTh that increases in a large number, be expanded to for five generations, use quick adenovirus infection titre (TCID50) detection kit measure after a large amount of amplifications the titre of recombinant adenovirus, titre is 2.5 * 10 ⁹IU/L confirms successfully to pack out a large amount of Ad5F35-CTLTh recombinant adenovirus vaccines.And confirm that genes of interest CTLTh all has expression at mRNA and protein level in incasing cells.

(4), use Ad5F35-CTLTh recombinant adenovirus transfection PERIPHERAL BLOOD MONONUCLEAR CELL (PBMCs), still keep activity more than 90% at 10 days PBMCs after the transfection, illustrate that recombinant adenovirus Ad5F35-CTLTh toxicity is extremely low; And behind the HCMV polyepitope vaccines stimulation PBMCs, can cause the amplification of HCMV specific CTL, thereby kill and wound special target cell.

The immune effect evaluation of the HCMV epitope adenovirus nucleic acid vaccine that Chinese population HLA of the present invention is special

(a), utilize EBV successfully to transform PBMCs, set up 2 strain lymphoblastoid cell lineses (lymphoblastoidcell lines, LCL);

(b), use the PBMCs that HCMV epitope adenovirus carrier bacterin Ad5F35-CTLTh stimulates the HCMV healthy carrier, the external efficient antigenic specificity CTL that increased;

(c), the HCMV CTL epi-position of Ad5F35-CTLTh coding can be offered by the processing of cell endogenous, thereby be killed and wounded by specific CTL; Ad5F35-CTLTh can cause the amplification of HCMV specific CTL, thereby kill and wound special target cell after stimulating PBMCs, and with imitating target than raising, the CTL cell killing activity of Ad5F35-CTLTh amplification strengthens;

(d), use ELISPOT experiment detection Ad5F35-CTLTh and stimulate HCMV healthy carrier's the PBMCs and the situation of antigenic specificity CTL secretion of gamma-IFN, found that Ad5F35-CTLTh can efficiently stimulate the PBMCs secretion of gamma-IFN, the CTL that twice stimulation amplification generates can produce higher a large amount of secretion of gamma-IFN.

In a word, the special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA of the present invention's preparation, 83 epitopes that comprise 15 kinds of HCMV virus proteins, cover 14 HLA I allele sites and 7 HLA II allele sites, the Chinese population coverage rate is up to 92.07% (75.49-99.99%); And have stronger immune effect, overcome previously weak, the immune scope limitation of the single antigen immune originality of HCMV vaccine shortcoming.

Description of drawings

The present invention is further described below in conjunction with accompanying drawing.

Fig. 1 is the present invention at the covering frequence of the Chinese population figure that predicts the outcome.The result shows, HLA combination of the present invention (A2, A24, A1, A3, A11, A68, B44, B7, A23, A26, B35, B38, B8, B27) in the Chinese population coverage rate up to 92.07% (75.49-99.99%).

Fig. 2 is HCMV polyepitope vaccines preparation flow figure of the present invention.Synthetic polypeptide epitope nucleotide fragments inserts shuttle vector pHMCMV5 by enzyme action, inserts vaccine carrier pAd5F35 then, changes incasing cells HEK293 over to, produces adenovirus vaccine Ad5F35-CTLTh.

Fig. 3 is HCMV polyepitope vaccines immune effect figure of the present invention.From healthy carrier JXX (HLAA2, A11, DR4, DR15) and ZNN (HLA A1, B44, B35, DR7, DR11) PBMCs and from the infection of body the PBMCs of Ad5F35-CTLTh (MOI 50: 1 and 25: 1) cultivated altogether 7 days than 2: 1 times imitating target, (MOI 50: 1) the x-roentgenization that infects with Ad5F35-CTLTh stimulates common cultivation once more from the LCL of body cell then, carried out the killing activity test of external epitope specificity T cell in the 10th day, the LCL that stimulates with single peptide section (20 μ g/ml) is a target cell, and imitating the target ratio is 10: 1.The HCMV polyepitope vaccines can cause the amplification of HCMV specific CTL, thereby kill and wound special target cell after stimulating PBMCs.

The specific embodiment

Below in conjunction with specific embodiment the present invention is described in further detail, but not as a limitation of the invention.

The preparation of the special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA

1. the prediction of epi-position and definite

Utilize Chinese HLA I accumulation phenotypic frequency spatial prediction system's (" 2005 the 21st the 2nd phases of volume of IMMUNOLOGY KEY WORDS INDEX, P136), choosing 14 HLA I gene locis makes up, as shown in Figure 1, HCMV polyepitope vaccines Chinese population HLA coverage rate of the present invention is up to 92.07% (75.49-99.99%).Then at 15 kinds of antigen proteins (pp28, pp50, the pp65 of HCMV, pp150, pp71, gH, gB, IE-1, IE-2, US2, US3, US6, US11, UL16 chooses 83 epi-positions in UL18), wherein,, can control the formation of duplicating, prevent the HCMV disease of virus in theory in each stage at 7 of the Th epi-positions (table 2) of HLA II gene loci at 76 of the CTL epi-positions (table 1) of above-mentioned 14 HLA I gene locis; The SYFPEITHI software prediction of 83 selected series connection epi-positions divides number average more than 14 minutes, and is most between 20-30 divides, and shows equal can being offered by the combination of HLA molecular specificity; MAPPP software prediction mark is many more than 0.500, proves that the polypeptide that forms can be degraded to corresponding epi-position in cell; Introduce the Kozak rule in the vaccine nucleotide sequence, add 6 His appraisement labels, then with the vaccine nucleotide sequence input DNAMAN software that obtains, the restriction enzyme site of having determined the HCMV series connection epi-position nucleotide sequence two ends that covering Chinese population HLA is special is Nhe I and Not I restriction enzyme site, can insert the multiple clone site of shuttle vector pHMCMV5 carrier.

HCMV CTL epi-position and prediction that table 1HLA I gene loci limits

The Th epi-position and the SYFPEITHI mark of the restriction of table 2HLA-II quasi-molecule

2. the polyepitope vaccines full-length gene is synthetic

According to the epi-position of selecting, adopt the synthetic HCMV polyepitope vaccines full-length gene CTLTh of gene splicing by overlap extension (SOEing) and round pcr.

(1), gene order is analyzed, it is synthetic respectively that gene is divided into 3 sections (A, B, C), and 112 forward and reverse primers have been synthesized in design by software, odd number is a forward primer, even numbers is a reverse primer, and other designs a primer and changes restriction enzyme site, 5 of sequencing primers.

(2), the synthetic pcr amplification of gene then, every fragment gene is synthetic all to use the secondary amplification.The A fragment gene is divided into two sections and synthesizes: A-1 (primer A1 to A18), A-2 (primer A19 to A38); The B fragment gene is divided into two sections and synthesizes: B-1 (primer B1 to B28), B-2 (primer B29 to B56); The C fragment gene synthesizes (primer C1 to C18).Amplification condition synthesizes example with A fragment gene A-1 fragment:

Contain in the 50ul reaction system of amplification for the first time: template (each 2pmol of each primer of A1 to A18), forward primer (A1) and each 5pmol of reverse primer (A18), PFU enzyme (2.5U/ul) 1ul, 10mM dNTP 1ul, 10 * buffer5ul, water complements to 50ul; Reaction condition: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, 25 circulations.

Contain in the 50ul reaction system of amplification for the second time: 2ul template (the PCR product increases for the first time), forward primer (A1) and each 10pmol of reverse primer (A18), PFU enzyme (2.5U/ul) 1ul, 10mM dNTP 1ul, 10 * buffer 5ul, water complements to 50ul; Reaction condition: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 25 circulations.

The amplification PCR products electrophoresis is cut glue purification for the second time, and other fragment amplifications finally obtain 5 genetic fragments by that analogy, and numbering is respectively A-1, A-2, B-1, B-2, C.

(3), the splicing full-length gene, with splicing A segment base because example:

Contain in the pcr amplification system of 50ul: 30ng template (genetic fragment A-1 and A-2), forward primer (A1) and reverse primer (A38), PFU enzyme (2.5U/ul) 1ul, 10mM dNTP 1ul, 10 * buffer 5ul, water complements to 50ul; Reaction condition: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 25 circulations.

By that analogy, finally pass through overlap extension PCR method with A, B, C three fragment genes are spliced into full-length gene, and recovery purification end adds the A rear clone and goes in the T carrier sequence verification (sequence is shown in SEQ ID No.1).Wherein linked system is: T carrier 30 as one kind ng, and genes of interest 60ng, ligase (5U/ul) 0.5ul, Buffer 1.5ul supplies water to 15ul, and 22 ℃ connect 1 hour, transform to get final product.

3. the preparation of adenovirus vaccine carrier

As shown in Figure 2, with Nhe I/Not I double digestion synthetic CTLTh fragment is gone into shuttle vector pHMCMV5 carrier from the T carrier cloning, make up shuttle vector pHMCMV5-CTLTh, identify shuttle vector pHMCMV5-CTLTh with Nhe I/Not I double digestion and I-CeuI/PI-SceI double digestion; Utilize I-CeuI/PI-SceI double digestion and extracorporeal recombination that the expression cassette that comprises CMV promoter, genes of interest and SV40 polyA tail on the shuttle vector is inserted adenovirus expression carrier pAd5F35, make up recombinant adenovirus vaccine expression vector pAd5F35-CTLTh, use I-CeuI/PI-SceI double digestion and XhoI enzyme action and identify recombinant adenoviral expressing vector pAd5F35-CTLTh.

4. the virus of adenovirus vaccine packing and a large amount of amplification

Use Pac I linearisation recombinant adenovirus vaccine expression vector pAd5F35-CTLTh, produce the ITR end, utilize liposome 2000 transfection HEK293, packing Ad5F35-CTLTh recombinant adenovirus: to the 60mm culture dish, cell reaches the fusion of 70-90% during transfection with suitable cell density inoculation HEK293 cell; Obtain solution 1 (240ul opti-MEM+10ul liposome 2000, cumulative volume 250ul, incubation 5min under the room temperature) and solution 2 (X ul opti-MEM+4ug Pac I enzyme action product, cumulative volume 250ul); Liposome 2000 is 2.5: 1 with the ratio of Pac I enzyme action product D NA, solution 1 is mixed room temperature underlying 20min with solution 2; The mixed liquor of solution 1 with solution 2 dropwise added in the culture dish, the wave and culture ware, mixing was hatched 4-6 hour in 37 ℃ of 5%CO2 incubators gently; After 6 hours, change the full culture medium that contains serum, in 37 ℃ of 5%CO2 incubators, continued to hatch 7-14 days observation of cell pathological changes effect (CPE); When CPE was obvious ,-80 ℃/37 ℃ froze the centrifugal recombinant adenovirus of collecting repeatedly molten three times.

Use the HEK293 cell Ad5F35-CTLTh recombinant adenovirus vaccine that increases in a large number: at 75cm ²Add 10ml 5%DMEM in the culture bottle and cultivate 5 * 10 ⁶293 cells; Get the virus preservation liquid that 0.5ul increases first, add 5%DMEM to 1ml, mixing dilutes the MOI value that obtains like this and is about 5; Remove culture fluid, carefully add viral mixed liquor, be sure not to destroy cell monolayer, cross slowly rocks 3 times, cultivates 90 minutes in 37 ℃ of 5%CO2 incubators, adds 9ml 5%DMEM again; Cultivated again 72 hours, at this moment in 10ml solution nearly 5 * 10 ⁹～5 * 10 ¹⁰Individual virion carries out MOI and measures to estimate virion;-80 ℃/37 ℃ multigelations 3 times change in the aseptic centrifuge tube of 15ml, centrifugal 10 minutes of 1500rpm, and it is frozen in-80 ℃ to collect supernatant; 3 175cm ²Respectively add 10 in the culture bottle ⁷293 cells are cultivated; 3ml cell pyrolysis liquid supernatant is added among the 12ml 5%DMEM, and mixing is removed cell culture fluid, every bottle of careful 5ml mixed liquor that adds, cross slowly rocks mixing 3 times, cultivates 90 minutes in 37 ℃ of 5%CO2 incubators, this moment, the MOI value was about 25, added 5%DMEM to 30ml; Cultivated 48～72 hours, this moment, 10ml culture fluid virus quantity was about 3 * 10 again ¹⁰～3 * 10 ¹¹Centrifugal 5 minutes collecting cells of horizontal centrifuge 1500rpm are abandoned supernatant, add the solution re-suspended cell of 1/10 initial volume.-80 ℃/37 ℃ freeze thawing 3 times with maximum rate sedimentation cell fragment, are collected supernatant on the desk centrifuge, carry out titration of virus.

The titre of using the recombinant adenovirus Ad5F35-CTLTh after quick adenovirus infection titre (TCID50) detection kit mensuration increases in a large number is 2.5 * 10 ⁹IU/L,

5. the evaluation of adenovirus vaccine

Use recombinant adenovirus Ad5F35-CTLTh transfection PBMCs, collected cell in the 5th, 7 days in transfection and carry out the cellular immunization chemical experiment, recombinant adenovirus Ad5F35-CTLTh all has expression at after birth, the endochylema of PBMCs; Expect that in the 1st, 3,5,7,10 days application station of transfection blue dyeing observes the cytoactive of PBMCs respectively simultaneously, still keep activity more than 90%, illustrate that recombinant adenovirus Ad5F35-CTLTh toxicity is extremely low at 10 days PBMCs after the transfection.

6. the functional checking of adenovirus vaccine

(1), utilize EBV successfully to transform PBMCs, set up 2 strain lymphoblastoid cell lineses (lymphoblastoid celllines, LCL); With the inductive CTL cell of the special peptide section of HCMV is the effector lymphocyte, the LCL cell that infects 16-18 hour HLA coupling with Ad5F35-CTLTh is a target cell, carry out lethal experiment, the HCMV CTL epi-position that found that the Ad5F35-CTLTh coding can be offered by the processing of LCL cell endogenous, thereby is killed and wounded by the HCMV specific CTL.

(2), (Ad5F35-CTLTh stimulates PBMCs with the effector lymphocyte, form the HCMV Specific CTL Cells) with target cell (bag by the special peptide section of HCMV from body LCL cell) immixture, after can finding that Ad5F35-CTLTh stimulates PBMCs, can cause the amplification of HCMV specific CTL, thereby kill and wound special target cell, and with imitating target than raising, the CTL cell killing activity of Ad5F35-CTLTh amplification strengthens;

(4), use ELISPOT experiment detection Ad5F35-CTLTh and stimulate HCMV healthy carrier's the PBMCs and the situation of antigenic specificity CTL secretion of gamma-IFN, found that Ad5F35-CTLTh can efficiently stimulate the PBMCs secretion of gamma-IFN, the CTL that twice stimulation amplification generates can produce higher a large amount of secretion of gamma-IFN.

Sequence table

＜110〉Shandong Qilu Hospital

＜120〉the special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA

<141>2009-6-11

<160>1

<170>PatentIn?version?3.3

<210>1

<211>2475

<212>DNA

<213>Human?cytomegalovirus

<400>1

gctagcgcca?ccatgaacct?ggtgcccatg?gtggctacgg?ttatcctggc?ccgcaacctg 60

gtgcccatgt?tgatgaacgg?gcagcagatc?ttcctgatgc?tgaacatccc?cagcatcaac 120

gtgcgcatct?tcgccgaatt?ggaaggcgta?gcgctcttct?ttttcgatat?cgacttggtc 180

ttagaggaga?ctagtgtgat?gctggtcctg?gcagaactcg?tcaaacagat?ttctctctta 240

agtgagttct?gtcgggtgac?catgtacggg?ggcatctctc?tcttatgttt?gcagaatgcc 300

ttagatatct?taattatcta?cacccgcaat?catgaggtgg?cgctggtcaa?cgccgtcaac 360

aagctcctca?tcgaggactt?tgatatctac?gtgaggatct?ggtgcctggt?agtctgcgtt 420

aacctctttc?cctatctggt?gtccgcctac?atcaaccggg?cgctggcgca?aatcatcctc 480

tttgatggac?acgatctgct?tctgctatat?cccacagccg?tggacctgtc?tatgatgtgg 540

atgcggtttt?tcgtgctgct?catcgacccc?acgtcggggc?tgaccctgct?cgtatatctg 600

ttctctctgc?tgctcaattg?cgccgtcacc?aaactgacct?tggcgagaga?catcgtgttg 660

gtcacaatgt?ggtgtctgac?gttgtttgtg?actcttttcg?atgaacctcc?gcccttggtg 720

ttcaccagcc?agtatcgcat?ccagggcaag?cttcagtacg?atcccgtggc?tgcgctcttc 780

gtgtacgcgc?tgccgctcaa?gatgctgcta?tatccgagac?cgcccggatc?cgggctcgag 840

tacatcgtgc?agatccagaa?tgctttcttg?tatttatgtt?gcgggattac?cctgcagtac 900

attctggggg?ccgatcctct?ggtgtatgtg?actgtggact?gtaacttggt?atatctgttc 960

tctctggttg?ttctttacag?cgagcacccc?accttcacca?gccagtatta?tcgcatccag 1020

ggcaagcttg?agtacatagg?tgaccagtac?gtcaaggtgt?acgttacgga?gcacgacacg 1080

ctgttgtacc?gtggcgatcc?cttcgacaaa?aattacgcca?tcatcggcat?ctatctgctc 1140

tacaataccg?acttcagggt?actggaactt?atcaaggagc?acatgctgaa?aaaatattgt 1200

gtggaaacaa?tgtgtaatga?gtacaccacc?gtttacccac?cgtcgtctac?ggccaaaacg 1260

gtacgcagtc?actgcgtgtc?aaaaacgctg?ctcaattgcg?ccgtcaccaa?aatcatgcgc 1320

gaattcaact?cgtacaagaa?aatgactgca?acttttctgt?ctaagggtat?caaacacgag 1380

ggcttagtca?aactggtgga?accgtgtgcg?cgcgtctact?tgctgctgca?gcgcgggcct 1440

cagtacagac?ttaacaccta?cgcattggta?tccaaactcg?tacgcctggt?ctacatactt 1500

tctaaaaagt?tggggggtgc?actgcaggct?aagggtccca?tttcggggca?cgtgctgaaa 1560

gctacggttc?agggtcagaa?tctgaagttc?gtgtttccca?ccaaggacgt?ggcactgcgg 1620

gtgacgtcga?cgccggttca?gggaaggcag?gaattcttct?gggacgccaa?cgacatctac 1680

tccgaagcgc?tggaccctca?cgcatttcgt?gaaaacacca?cccagtgcac?ctacacggaa 1740

aacggtagtt?ttgtagccgg?ttacacgccc?cgcgtcaccg?gcggcggcgc?catggccccg 1800

gtcgcgggta?gtatgcctga?attaaaggcc?cgtgctaaaa?aggatgaact?taaggcgcgt 1860

gaccacctgg?ctgtgctatc?gccctgggct?ccgacggcgc?cgttgtggcc?gcgcgaacgc 1920

gcgtgggccc?tctctccgcg?aactcactac?ctcatgcttg?cttatgccca?gaaaatattt 1980

aagattttgt?acgtcaaggt?gtacctggag?tccttctttc?ccaccaagga?cgtggcactg 2040

cccaccttca?ccagccagta?tcgcatccag?ggcaagcttc?agattaaggt?tcgagtggac 2100

atggtgagga?gaaagatgat?gtatatgtgc?tacccgctca?agatgctgaa?catccccagc 2160

atcaacgtgc?accactacga?gcccgacgtc?tactacacgt?cagcgttcgt?gtttcccacc 2220

aagaaggtgt?acctggagtc?cttctgcgag?gacgtgccct?ccggcaagat?aatcaaaccg 2280

ggcaagatct?cgcacatcat?gctggatgtg?gctcacccca?ccttcaccag?ccagtatcgc 2340

atccagggca?agcttgaggc?cggcatcctg?gcccgcaacc?tggtgcccat?ggtggctacg 2400

gtttaccagg?aattcttctg?ggacgccaac?gacatctacc?gcatcttcca?tcatcaccat 2460

caccattgaa?cgcgt 2475

Claims

1. special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA, form by human cytomegalic inclusion disease virus nucleotide tandem sequence CTLTh and adenovirus expression carrier pAd5F35, it is characterized in that, described human cytomegalic inclusion disease virus nucleotide tandem sequence CTLTh is the nucleotide sequence shown in the SEQ ID No.1, this nucleotide sequence comprises the nucleotide sequence of 83 epitopes of 15 kinds of encoding proteins of human cytomegalic inclusion disease virus, and this sequence covers 14 HLA I allele sites and 7 HLA II allele sites.

2. the special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of Chinese population HLA according to claim 1 is characterized in that, 15 kinds of encoding proteins of described human cytomegalic inclusion disease virus are meant pp28, pp50, pp65, pp150, pp71, gH, gB, IE-1, IE-2, US2, US3, US6, US11, UL16 and UL18; Described 14 HLA I allele sites are meant A2, A24, A1, A3, A11, A68, B44, B7, A23, A26, B35, B38, B8 and B27; Described 7 HLA II allele sites are meant HLA-DR1,3,4,7,11,13 and 15.

3. the preparation method of the special human cytomegalovirus Multi-epitope adenovirus nucleic acid vaccine of the described Chinese population HLA of claim 1, step comprises: (1), determine and prediction covers the special HCMV series connection epi-position of Chinese population HLA; (2), the special HCMV epitope adenovirus vector nucleic acid vaccine of preparation Chinese population HLA; It is characterized in that,

The concrete grammar of step (1) is:

(a), utilize Chinese HLA-I accumulation phenotypic frequency spatial prediction system, be A2 to bonded 14 the HLA gene locis of selected HCMV epi-position, A24, A1, A3, A11, A68, B44, B7, A23, A26, B35, B38, B8 and B27 combination are predicted;

(b), be pp28 at 15 kinds of antigen proteins of HCMV, pp50, pp65, pp150, pp71, gH, gB, IE-1, IE-2, US2, US3, US6, US11, UL16 chooses 83 epi-positions among the UL18, wherein at 76 of the CTL epi-positions of above-mentioned 14 HLA-I gene locis, at 7 of the Th epi-positions of HLA-II gene loci, with the formation of duplicating, prevent the HCMV disease in each stage control virus;

(c), use DNAMAN software, Kozak rule and PubMed Nucleotide NC_001347.Humanherpesvirus 5 strain AD169 HCMV genomes and draw the special HCMV polyepitope vaccines nucleotide sequence of covering Chinese population HLA, wherein added the Kozak sequence in origin or beginning, end has added 6 His sequence labels, and two ends have added Nhe I and Not I restriction enzyme site;

The concrete grammar of step (2) is:

(a), adopt gene splicing by overlap extension and the synthetic HCMV polyepitope vaccines epi-position full-length gene CTLTh of round pcr, its nucleotide sequence is shown in SEQ ID No.1;

(b), utilize homologous recombination technique, the CTLTh gene is inserted the Nhe I/Not I restriction enzyme site of shuttle vector pHMCMV5, make up shuttle vector pHMCMV5-CTLTh, use Nhe I/Not I restriction analysis and sequencing analysis and identify the purpose carrier;

(c), utilize I-CeuI/PI-SceI double digestion and extracorporeal recombination that the expression cassette that comprises CMV promoter, genes of interest, SV40polyA tail on the shuttle vector is inserted adenovirus expression carrier pAd5F35, make up recombinant adenoviral expressing vector Ad5F35-CTLTh, use analysis of Xho I enzyme action and I-Ceu I/PI-Sce I double digestion and sequencing analysis and identify the purpose carrier;

(d), use PacI linearisation recombinant adenovirus vaccine expression vector Ad5F35-CTLTh, with liposome 2000 transfection HEK293, packing Ad5F35-CTLTh recombinant adenovirus vaccine is waited to cultivate and was collected virus when the CPE effect occurring in 10 ± 1 days; Use the HEK293 cell Ad5F35-CTLTh recombinant adenovirus that increases in a large number, be expanded to for five generations, use the titre that quick adenovirus infection titre TCID50 detection kit is measured the recombinant adenovirus after a large amount of amplifications;

(e), use recombinant adenovirus Ad5F35-CTLTh transfection PBMCs, check recombinant adenovirus Ad5F35-CTLTh toxicity.