Comparative Study on the Immunogenicity between Hsp70 DNA Vaccine and Hsp65 DNA Vaccine in Human Mycobacterium Tuberculosis

The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 μg/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P<0.01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P<0.01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P>0.05); The contents of serum IFN-γ in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P<0.05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying.

DNA Vaccines Approach:From Concepts to Applications

DNA vaccines are the third generation vaccines based on purified plasmid preparations containing transgenes that encode antigenic/therapeutic proteins or peptides capable of triggering an immune response against a wide range of diseases. This vaccine platform presents several attributes that confer distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. Many aspects, such as antigen expression and especially vector design, are under study because of their great influence on immunogenicity and efficacy of DNA vaccines. In this regard, with the attempt of improving the efficiency of DNA vaccines, co-expression of stimulatory sequences and diverse vector delivery systems are being optimized. With this in mind, this review aims to giving a conceptual approach of DNA vaccines, explaining their mechanisms of action and listing the already licensed veterinary DNA vaccines presented in the market.

Effect of BNT162b2 mRNA COVID-19 vaccine on sperm morphokinetics and DNA integrity: A prospective observational study in Japan

Objective:To assess whether the coronavirus disease 2019(COVID-19)mRNA vaccine affects sperm morphokinetics using a computer-assisted semen analyzer and other semen parameters using a sperm chromatin structure assay.Methods:Healthy male volunteers in two Japanese clinics between May 2021 and December 2021 were prospectively analyzed.Participants donated sperm twice,two days apart,in the following phases:before vaccination,2 weeks after the first vaccine dose,and 2,4,and 12 weeks after the second dose.Basic sperm parameters,sperm motility characteristics,and the percentage of DNA-damaged sperm were compared among the different phases.Results:Ninety-six semen samples from ten volunteers,who were vaccinated with the BNT162b2 mRNA vaccine,were evaluated.There were no significant differences between any phases in basic semen findings and parameters of the sperm chromatin structure assays.Regarding sperm motion characteristics,the average linear velocity,beat-cross frequency,and sperm motility index significantly decreased after the second vaccine dose(P=0.018,P=0.003,and P=0.027,respectively),with no significant differences between any two phases by post-hoc pairwise comparisons.Conclusions:After COVID-19 mRNA vaccination,while sperm motion characteristics might fluctuate,no apparent deterioration of basic sperm parameters or sperm DNA integrity was observed.Given the adverse effects of COVID-19 on sperm,our findings suggest that there might be no reason to refrain from vaccination for healthy individuals.

Recent advances in DNA vaccine of hepatitis virus

Nucleic acid vaccine or DNA vaccine is a hopeful vac- cine to prevent and treat viral hepatitis. Problems exist in different DNA vaccines for HBV or HCV. Optimal animal model should be established study vaccine against hepatitis. Apart from the strategy to enhance the efficiency of DNA vaccine, combined use of cytokines or chemokines, different routes of inocu- lation, design of optimal vector, ISS insertion in the plasmid vectors, etc to enhance the efficiency of DNA vaccine are reviewed.

Enhancement Effect of CpG DNA on the Somatostatin DNA Vaccine in Mice

To study the immune effect of CpGDNA on somatostatin （SS） DNA vaccine, the 20-day-old experimental mice were immunized with 20 lag SS eukaryotic expression plasmid pES/2SS with different adjuvants in equal dose, such as the synthetic CpG-ODN, the pE-CpG plasmid, E. coli DNA and the crude liposome. A booster was given two weeks later. The results showed that the body weight gain of female mice in the SS immunized group was higher than that of the control （P 〈0.05）. The levels of antibodies against SS, IgG2a/IgG1, spleen lymphocyte proliferation activity and the concentrations of GH and IGF-Ⅰ in the DNA vaccine groups combined with CpGDNA were significantly increased compared to that of the group immunized with DNA vaccine alone. All these suggested the recombinant SS expression plasmid can stimulate animals to produce antibodies against SS, and CpGDNA adjuvant can enhance the immune effect of DNA vaccine against SS and influence the concentration of GH and IGF-Ⅰ .

Humoral and cellular immunogenecity of DNA vaccine based on hepatitis B core gene in rhesus monkeys

INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].

The past, current and future trends in DNA vaccine immunisations

This review focuses on DNA vaccines, denoting the last two decades since the early substantiation of preclinical protection was published in Science in 1993 by Ulmer et al. In spite of being safely administered and easily engineered and manufactured DNA vaccine, it holds the future prospects of immunization by inducing potent cellular immune responses against infectious and non-infectious diseases. It is well documented that injection of DNA plasmid encoding a desired gene of interest can result in the subsequent expression of its products and lead to the induction of an immune response within a host. This is pertinent to prophylactic and therapeutic vaccination approach when the peculiar gene produces a protective epitope from a pathogen. The recent studies demonstrated by a number of research centers showed that these immune responses evoke protective immunity against several infectious diseases and cancers, which provides adequate support for the use of this approach. We attempt in this review to provide an informative and unbiased overview of the general principles and concept of DNA vaccines technology with a summary of a novel approach to the DNA vaccine, present investigations that describe the mechanism(s) of protective immunity provoked by DNA immunization and to highlight the advantages and disadvantages of DNA immunisation.

Protective effect of DNA vaccine with the gene encoding 55kDa antigen fragment against Pneumocystis carinii in mice

Objective:To evaluate the protective effect of DNA vaccine with the gene encoding 55kDa antigen fragment of Pneumocystis carinii(P.carina) against P.carina in mice.Methods:The fragment of the antigen within p55(p55-582) was cloned.Then recombinant plasmid was constructed based on the eukaryotic expression vector pcDNA3.1(+).BALB/c mice were used as experimental models to examine the immunogenicity of pcDNA3.1(+)-p55-582.ELBA and RTPCR were used to evaluate the role of this kind of DNA vaccine.Results:The results of western blot indicated that the recombinant DNA[pcDNA3.1(+)-p55-582]could be expressed correctly and had antigenicity in transfected COS-7 cells.ELBA and RT-PCR showed that pcDNA3.1(+)- p55-582 elicited antibody production,stimulated lymphocyte proliferation and provided partial protection by reducing the P.carina burden.Conclusions:The data demonstrate that pcDNA3.1(+)-p55-582 might be potent vaccination that can afford the partial protection for the immunized animals.

Construction and Expression of DNA Vaccine pIRES-Sj97-Sj14-Sj26 and Its Immunogenicity in Mice

To find a new preventive strategy for the infection of Schistosoma japonica, plasmid pIRES-Sj97-Sj 14-Sj26 that contains fatty binding protein （Sj 14）, GST （Sj26） and paramyocin （Sj97） that are expressed on the membrane, was constructed. RT-PCR was used to detect the expression of Sj 14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, the indirect immunofluorescent test was employed for the detection of the expression of trans-membrane Sj26 after the plasmid was transfected into Hela cells. Fifty BALB/c mice were randomly divided into 5 groups and plRES-Sj97-SjI4-Sj26 plasmid DNA, plRES-Sj 14-Sj26 plasmid DNA, plRES-Sj26 plasmid DNA, plRES blank vector and normal saline were respectively injected into the quadriceps muscles of thigh Eight weeks after the immunization the mice were killed and significantly higher level of IgG was detected in the plRES-Sj97-Sj 14-Sj26 group as compared with the plRES blank vector, normal saline and plRES-Sj26 groups （P〈 0.01） and the plRES-Sj 14-Sj26（P〈0.05）. Single splenocyte suspension was prepared to detected the level of IFN-T by ELISA and the lymphocyte stimulating index （SI） by MTT. SI was significantly higher of in the plRES-Sj97-Sj 14-Sj26 group than in other groups （P〈 0.01）, while the IFN-T level was significantly higher the plRES-Sj97-Sj 14-Sj26 group than in plRES blank vector and normal saline groups （P〈0.01）, but no significant differences were found when compared with plRES-Sj 14-Sj26 and plRES-Sj26 groups. Flow cytometery showed that the percentages of CD4＋ and CD8＋ T cells were much higher in the plRES-Sj97-Sj 14-Sj26 group （P〈 0.01, P〈0.05）. It was concluded that plRES-Sj97-Sj 14-Sj26 vaccine may induce stronger immune response in BALB/c mice.

Phase Ⅱb trial of in vivo electroporation mediated dualplasmid hepatitis B virus DNA vaccine in chronic hepatitis B patients under lamivudine therapy

AIM To assess the efficacy and safety of in vivo electroporation(EP)-mediated dual-plasmid hepatitis B virus(HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine(LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine(vaccine group, n = 109) or LAM + placebo(control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12(start of treatment with vaccine or placebo, SOT), 16, 24, and 36(end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, morepatients had a decrease in HBV DNA > 2 log10 IU/m L in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir(ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeA g seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy.

Specific cellular immunity and antitumor responses in C57BL/6 mice induced by DNA vaccine encoding murine AFP

BACKGROUND: The inoculation of plasmid DNA encod-ing tumor-associated antigens is a novel and powerful stra-tegy for antitumor vaccination. This study was designed toconstruct the DNA vaccine of mouse AFP and to observethe specific cellular immunologic responses and the antitu-mor responses in mice induced by this vaccine.METHODS: The murine AFP gene was amplified by RT-PCR from total RNA extracted from Hepal-6 cells andcloned into the vector pcDNA3.1 to construct pmAFP.The DNA vaccine was identified by restriction enzymeanalysis, sequencing and expression. EL-4 ( mAFP) wasdeveloped by stable transfection of EL-4 cells with pmAFP.The frequency of cells producing IFN-γ in splenocytes har-vested from the mice immunized with the DNA vaccine byintramuscular injection was measured by enzyme linkedimmunospot (ELISPOT). The mice immunized with theDNA vaccine were inoculated with EL-4 (mAFP) cells inback to observe the inhibitory effect of the immunizationon tumor. On the other hand, blood samples were collect-ed from the immunized mice to check the functions of theliver and kidney.RESULTS: The murine AFP gene was successfully clonedby RT-PCR. Results from restriction enzyme analysis, se-quencing and expression showed that the DNA vaccine wassuccessfully constructed. The expression of mAFP mRNAin EL-4 (mAFP) was confirmed by RT-PCR. The resultsof ELISPOT showed that the number of IFN-γ-producingcells of the pmAFP vaccine group was significantly higherthan that of other groups (P <0.01). The tumor volume inthe pmAFP vaccine group (1042. 42 ± 123. 71 mm3 ) wassignificantly smaller than that in other groups (P<0.01).The function of mouse liver and kidney in each group wasunchanged.CONCLUSION: The successfully constructed DNA vaccineof AFP can induce specific cellular immunologic responsesand significant antitumor reponses in mouse and has no im-pact on the function of mouse liver and kindey.

Immunogenicity and immunizing protection effect of GAMA gene DNA vaccine on Plasmodium berghei

Objective: To explore the effect of immunogenicity and immunizing protection of GAMA gene DNA vaccine, which was related with merozoite, ookinete and sporozoite invasion. Methods:Gene fragments were obtained using PCR technique and eukaryotic expression vector(containing immunostimulatory sequence) was built. BALB/c mice were divided into PBS control group, empty vector control group and study group and were immunized at week 0, 3 and 6 respectively. Blood was collected 2 weeks after each immunization and serum was separated to detect the Ig G, Ig G1 and Ig G2 a levels. Spleen of mice was obtained for preparation of splenic mononuclear cell and the cytokine IL-4 and IFN-αlevels were detected. Indirect immunofluorescence and western blot were employed to verify the specificity of antiserum. Sporozoite and merozoite invasion were used respectively to detect the immune protective effect 2 weeks after the third immunization. Ookinete conversion rate in vitro and oocyst numbers of mosquito stomach were observed to evaluate the transmission-blocking levels. Results: In GAMA DNA vaccine group: antiserum could be combined with recombinant protein specifically and green fluorescence signals of merozoite, ookinete and sporozoite were observable, while specific fragments and fluorescence signals were not observable in empty vector group. Compared with control group, specific Ig G in DNA vaccine immunity group significantly increased(P<0.01), and Ig G1 and Ig G2 a all increased(P<0.01). IL-4, IFN-αcontent in study group significantly increased, compared with control group(P<0.01). GAMA DNA vaccine immunity could not obviously block the erythrocyte-stage infection(caused by sporozoite invasion); compared with control group, liver worm load was slightly reduced(P<0.05), and antiserum ookinete numbers(cultured in vitro) had no significant difference with oocyst numbers of mosquito stomach in DNA vaccine group. Conclusions:GAMA has good antigenicity, which could stimulate the body to produce specific immune responses; while DNA vaccine immunity could not play a good protective effect, the effect of which is only limited to the slight reduction of liver worm load, and has no obvious erythrocytestage protective effect and transmission-blocking effect. Therefore, trying other immunization strategies for further research on the value of GAMA(as multi-stage antigen vaccine and multistage combined vaccine components of the life-cycle of plasmodium) is necessary.

The Protection Efficacity of DNA Vaccine Encoding Hemagglutinin of H5 Subtype Avian Influenza Virus

The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10, 40, 70, 100 and 150 μg groups immunized with pCIHA5 were 12.5 (1/8), 58.3 (7/12), 72.7 (8/11), 50.0 (6/12) and 66.7% (8/12), respectively. The protective rates in 5, 20, 35 and 50 μg groups were 145.5 (5/11), 58.3 (7/12), 58.3 (7/12) and 91.7% (11/12), respectively. The 70, 100 and 5 μg groups have virus shedding of 1/8, 2/6 and 1/5. Though the inactived oil-emulsion vaccine has high HI antibody titers and 100% protective rate, the AGP antibody could be detected after vaccination. Results show that the pCIHA5 is fit to boost by intramuscular injection. This would be useful to the study on gene engineering vaccine of avian influenza virus.

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease,caused by the lymphocystis disease virus (LCDV),is a significant worldwide problem in fish industry causing substantial economic losses.In this study,we aimed to develop the DNA vaccine against LCDV,using DNA vaccination technology.We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate.The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line.The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR,RT-PCR and fluorescent microscopy.Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver,6 and 25 days after vaccination.The vaccine plasmids disappeared 100 d post-vaccination.Fluorescent microscopy revealed green fluorescence in the injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver of fish 48 h post-vaccination,green fluorescence did not appear in the control treated tissue.Green fluorescence became weak at 60 days post-vaccination.RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6-50 days post-vaccination.These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish.The antigen would therefore potentially initiate a specific immune response.The plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated.The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD.Further studies are required for the development and application of this promising DNA vaccine.

Studies on DNA Vaccine of the Conservative Region of Gene Encoding the Male-Specific Gynecophoral Canal Protein of Schistosoma japonicum (Chinese strain)

In order to observe the protective effect induced by vaccinating animals with the DNA vaccine of Sex-specific expression gene of Schistosoma, A 868 bp cDNA fragment amplified by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNA was cloned into the eukaryotic expression vector pcDNA3 and the recombinant eukaryotic expression vector pcDNA3-SjGCP1 was directly injected into BALB/c mice intramuscularly 3 times with the interval of 3 weeks .Both the vaccinated and control group of mice were challenged with 40 cercariae of Sj 5 weeks after last injection and perfused 7 weeks post-challenge. The worm and egg reduction rate got from vaccinated mice was 32.4% and 46.9% respectively. The result indicated that pcDNA3-SjGCP1 DNA vaccine induces the significant protection in animal against Schistosoma japonicum infection.

HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

Using human papillomavirus type 16 （HPV16） E7 as an antigen and Heat Shock Protein 70 as adjuvant, we constructed a DNA vaccine by linking HSP70 gene to E7^C91G; gene. Mice, after being immunized with E7^C91G;-HSP70, E7^C91G/HSP70, E7^C91G, and wild E7 DNA vaccines respectively, produced E7 specific CD8^＋ T-cell precursor frequencies of 280.33±2.52, 144.34±4.04, 164.34±5.13 and 82.33±3.51 respectively within every 1 × 10^5 mouse splenocytes. This proves that E7^C91G-HSP70 fusion vaccine can significantly enhance the E7 specific cellular immunity within the mice body（p〈0. 01）. After being immunized with E7^C91G-HSP70 fusion vaccine, tumor-bearing mice of the group being treated have significantly longer latency and survival periods, comparing with other three categories of E7 vaccines. Experiment shows that this vaccine has a significant effect on enhancing E7 positive tumor-treatment within mice body. After being immunized with E7^C91G-HSP70 vaccine, there were no pathological changes found in livers, kidneys and spleens of the mice, which proves that the vaccine is quite safe. After all, E7^C91G-HSP70 fusion vaccine has a much stronger tumor- treatment effect than thai of wild type E7 DNA vaccine.

Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses

Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.

Co-administration of Interleukin-2 Enhances Cellular and Humoral Immune Responses to HIV Vaccine DNA Prime/MVA Boost Regime

Interleukine-2(IL-2) is a growth factor for antigen-stimulated T lymphocytes and is responsible for ~T-cell clonal expansion after antigen recognition. It has been demonstrated that DNA vaccine-elicited immune responses in mice could be augmented substantially by using either an IL-2 protein or a plasmid expressing ~IL-2. Twenty mice, divided into four experimental groups, were immunized with: (1) sham plasmid; ^(2) HIV-1 DNA vaccine alone; (3) HIV-1 DNA vaccine and IL-2 protein; or (4) HIV-1 DNA vaccine and IL-2 plasmid, separately. All the groups were immunized 3 times at a 2-week interval. Fourteen days after the last DNA vaccine injection, recombinant MVA was injected into all the mice except those in group 1. ELISA and ELISPOT were employed to investigate the effect of IL-2 on DNA vaccine immune responses. The obtained results strongly indicate that the efficacy of HIV vaccine can be enhanced by co-administration of a plasmid encoding IL-2.

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

H5N1 鸟的流行性感冒病毒(A/chicken/Hubei/489/2004 ) 的 M 和 NP 基因被 RT-PCR 从病毒的 RNA 放大，并且分别地克隆向量进 pMD18-T。包含 M 基因(pHM6-m ) 或 NP 基因(pHM6-np ) 的表示 plasmid 然后被把 M 或 NP 基因插入到 pHM6 优核质表示向量构造；构造 plasmid 然后被定序。32 只 BALB/c 老鼠(6-week-old ) 在随机被划分成四个组。三组 BALB/c 老鼠被接种一次有 plasmid pHM6-m， plasmid pHM6-np 的 30 渭 g 或 plasmid pHM6-m (15 渭 g ) 和 pHM6-np (15 渭 g ) 的混合的任何一个 30 渭 g 的肌内的线路分别地。老鼠的一个另外的组作为控制与 100 渭 l PBS 被注射。二个星期以后，所有老鼠与相应 H5N1 鸟的流行性感冒病毒被质问，并且在下列 12 天内观察了。在 pHM6-m 组， pHM6-np 组和混合 plasmids 组的老鼠的幸存率分别地是 62.5% ， 25.0% 和 50.0% 。结果证明有效保护能被 pHM6-m 或 pHM6-np 提供，但是 pHM6-m 比 pHM6-np 提供了更好保护的效果。关键词 H5N1 流行性感冒病毒 - M 基因 - NP 基因 - 克隆 - DNA 疫苗的 CLC 数字 S852.65 基础条款：国家基本科学才能训练资助(NSFC J0630648 )

Effects of Cytokine IL-18 Gene on Antibody Production Induced by Ag85A DNA Vaccine

Objective:To investigate the effects of plasmid containing human IL-18 gene on the humoral immune response of mice immunized by Ag85A DNA vaccines of Mycobacterium tuberculosis H 37R v strain.Methods: Human IL-18 cDNA was amplified from RNA of peripheral blood mononuclear cells(PBMCs)by RT-PCR and cloned into the pGEM-TEasy vector.After sequencing IL-18 gene was subcloned into the the sites of BamH Ⅰ and EcoR Ⅰ digestion of pcDNA3.1. BALB/c mice were injected intramuscularly with eukaryotic expression plasmid pcIL18,together with MTB pcAg85A DNA vaccines.The same immunization was repeated three times at intervals of two weeks.Mouse sera were collected at two weeks after the each injection.The titers of anti-Ag85A antibody were detected by ELISA. Results:IL-18 cDNA was amplified successfully from RNA of human PBMCs by RT-PCR and the result of sequencing was correct.The IL-18 gene was correctly inserted into the vector pcDNA3.1, which was confirmed with BamHⅠ and EcoRⅠ digestion analysis. The positive plasmid was called pcIL18.After being immunized with DNA vaccines,the titers of antibody obtained from mice being immunized by pcAg85A combining with pcIL18 were superior to mice immunized by pcAg85A independently.Conclusion:Combination of IL-18 gene with MTB pcAg85A DNA vaccine could observably enhance the humoral immune responses to pcAg85A.It remains further investigated whether IL-18 gene plus MTB pcAg85A DNA vaccine could markedly induce the cellular mediated immune response to Ag85A or not.