【作者】 白靓；

【导师】 关铭；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 流感是一种由流感病毒引发的传染病,患者以发热、粘膜充血、急性呼吸道症状为主。该病发病率高,破坏性强,从二十世纪三十年代人类发明流感疫苗至今,每年仍有25～50万人死于流感。季节性流感以H1、H3亚型为主,而禽流感H5、H7亚型也不断出现人类感染病例。流感病毒不断变异,随时都有可能进化成为大流行流感毒株,世界上很多国家都在研发新型流感疫苗以应对多种亚型流感大流行。本研究利用生物信息学方法、免疫学知识,对H1、H3亚型流感病毒的T、B细胞表位进行预测与筛选,获得CTL表位3条:H1HA545-553、H3HA546-554、H3NP189-197,B和Th细胞表位4条:H1HA142~156、H1HA212~226、H3HA247~261、H3HA287~301。结合已发表的表位,设计、合成CTL、B/Th多表位表达盒。以H5HA、H7HA、H1NP、多表位表达盒为主要目的基因,pVAX1为载体构建用来预防H5、H7、H1、H3亚型流感的多表位核酸疫苗。将所构建的疫苗载体转染BHK细胞,通过RT-PCR和IFA验证核酸疫苗抗原成分可以在真核细胞中成功表达。利用小鼠作为哺乳动物模型,对所构建的复合多表位核酸疫苗免疫原性进行研究,表明构建的核酸疫苗在诱导细胞免疫及体液免疫方面具有显著优势。本实验的创新之处在于:1)依靠H5、H7亚型流感主要抗原成分,结合H1、H3抗原的功能表位构建了一种新型复合多表位核酸疫苗,目的是通过疫苗接种来预防H5、H7、H1、H3多种亚型流感病毒;2)预测多条H1、H3亚型流感功能表位,并将表位设计成能够发挥功能的多表位表达盒;3)通过小鼠实验免疫评价构建核酸疫苗的免疫原性。更多还原

【Abstract】 Influenza is a highly infectious disease caused by frequently mutating influenza viruses. The major symptom are fever, mucous congestion and acute respiratory symptoms. This disease has a high incidence and characteristics of strong destruction. Thirties the twentieth century, mankind invented the flu vaccine. Up to now, 25 to 50 million people die annually worldwide of influenza epidemic outbreaks. According to the WHO, every year influenza viruses infected 10-20% of the total population. The greatest threat to mankind are primarily H1/H3/H5/H7 subtype.Influenza vaccine can prevent infection and spread of the virus. The traditional vaccines proved safe and effective clinically include whole inactivated virus vaccine, live attenuated vaccine, split vaccine and subunit vaccine. But there are still some disadvantages can not be avoided, such as low level-immunization, high production costs and lack of immune monitoring and the key is that traditional vaccines can not effectively deal with the antigenic drift or antigen variation. So developing new influenza vaccine is the research focus in various countries all the time. DNA vaccine is a new technology originated in the nineties the twentieth century with its advantages stimulating the body to generate humoral and cellular immunity at the same time and it’s easily operate, build and safe. Pathogenic influenza virus antigens in the cells contains many conservative epitopes. To design and develop epitope-based multi-epitope vaccine, it can make up for the whole inactivated virus vaccine, attenuated live vaccine less than a single subtype of protection. Epitope is part of the immune system can generate immune response region, typically from 6-15 amino acid epitope or groups composed of carbon and water. It can be formed by the amino acid sequence of consecutive or discontinuous spatial three-dimensional structure of protein composition. Epitope vaccine based on defined epitopes, combined with some carriers such as the viral/ bacterial vectors or liposomes and virus-like particles.The body is immunitied after immunization against the infection. To epitope-based vaccines can efficiently use a variety of protective antigen of influenza viruses to reach the purpose of preventing a variety of virus subtypes. This study combined DNA vaccine and the advantages of epitope vaccines. We are researched on a multi-epitopes of influenza DNA vaccine. Details are as follows:(1) Using bioinformatics methods, immunization and screening knowledge predict- ed H1, H3 subtypes of influenza virus T, B cell epitopes. We designed and synthesized CTL, B/Th multi-epitope expression cassette each one.(2) This study was designed multi-CTL epitope expression cassette H5HA, H7HA series connected to the pVAX1 vector. Construction of H5, H7 influenza multi-epitope DNA vaccine. The B/Th multi-epitope gene expression cassette and H1NP series connected into the pVAX1 vector. Construction of NP multi-epitope DNA vaccine.(3) The vaccine vector was transfected into BHK cells.By RT-PCR and IFA verify the expression of DNA vaccine antigen component of the various activity.(4) Using mice as a mammalian model, the establishment of five immunization groups, two control group evaluate the ability which producted by complex multi-epitope DNA vaccine induced cellular immunity and humoral immune.1 Forecasting H1, H3 subtypes of influenza virus T, B epitope and designed multi-epitope expression cassette.(1) Forecasting H1, H3 subtypes of influenza virus T, B epitopeInfluenza virus HA is the main component of the virus surface antigen. It can stimulate body to produce protective immunity. It is important to influenza vaccine target. The NP, M1 protein that is sequence conservation, can induce CTL immune response and immune protection. Because of its conservative design and more universal influenza vaccine, it is an important target antigen target. We predict and screen the H1, H3 subtypes of influenza virus structural proteins HA, NP, M1 T-cell epitopes, B cell epitopes by bioinformatics methods. It is obtained that the three CTL epitopes: H1HA 545~553, H3HA546~554, H3NP189~197, B and Th cell epitope 4: H1HA142~156, H1HA212~226, H3HA247~261, H3HA287~301.(2) Multi-epitope expression cassette design.Previous studies have shown the four CTL epitopes: H1HA344~353, H3NP380~393, H1NP1~9, H1M12~12; B cell epitope: H1HA344~357, H3HA91~108. Combined with their own forecast table phase, we design multi-CTL epitope expression cassette and B/Th epitope expression cassette. Two types of multi-epitope expression cassette are connected in series form. We used the unique connection which connect epitope to anthor epitope that protect the functional form to play. In the two expression cassettes were added in the Igκ-chain signal sequence. It can promote the potential energy of complex multi-table well into the functioning of endoplasmic reticulum region. PADRE sequence to optimize the use and enhance immunity. We named more than two epitope expression cassette for the CTL ep and B/Th ep. The design of multi-epitope amino acid sequence expression cassette restore the nucleic acid sequence..2 Construction and identification of H5, H7 influenza multi-epitope DNA vaccine(1) Construction of H5, H7 influenza multi-epitope DNA vaccineWe cloned influenza virus H5HA, H7HA, NP gene by molecular biology techniques. And the corresponding genes were PCR transformation. This was for constructing DNA vaccine. DNA vaccine is based on pVAX1 as vector. Construction of pVAX1-H5HA, pVAX1-H7HA, pVAX1-NP, pVAX1-H5HA-H7HA. On this basis, the multi-epitope expression box B/Th ep sequence transformed into pVAX1-NP gene of the vector, and the NP gene co-expression vector named as pVAX1-NP-B/Th ep; the multi-epitope expression Box CTL ep sequence transformed into pVAX1-H5HA-H7HA carrier, CTL ep sequence in the middle of H5HA, H7HA and the vector named pVAX1-H5HA-CTL ep-H7HA.(2) Identification of H5, H7 influenza multi-epitope DNA vaccineBy restriction enzyme digestion and sequencing, it determin the constructed recombinant DNA vaccine including HA, NP, M1 sequence and nucleic acid sequence. This was selected strains of the same composition, consistent with the expected restrict- tion fragment sizes for further of the joint function of multi-epitope vaccine basis.3 Expression of H5, H7 influenza multi-epitope DNA vaccine in BHK cells.Using recombinant pVAX1-NP-B/Th ep, pVAX1-H5HA-CTL ep-H7HA transfer- cted BHK cells.We detected gene expression by RT-PCR and IFA. Through the different antibody tests,we found NP, CTL epitope expression cassette, B/Th multi-epitope expression cassette, H5HA, H7HA can be expressed in BHK cells, and it has activity. The results confirmed that the antigen components can be effectively expressed in eukaryotic cells.4.Study on immunogenicity of H5, H7 influenza multi-epitope DNA vaccine(1) Animal group, immune and samplingTake 6-8 weeks old female seventy BALB/c mice, ten in each group, divided into five immunization groups, two control group. Respectively, 0d, 21d, 35d, immunization three.We got rat ophthalmic artery blood ten days after the third immunization.It was prepared to serum antibody. After the mice were killed and blood. We prepared sterile whichever spleen spleen cell suspension for detecting cellular immunity indexes in.(2) Detection of H5, H7 multiple epitope DNA vaccine induced antibody and cellul- ar immune response.Proved by indirect ELISA epitope vaccine immunization can stimulate the expression of antibodies and immune antigen alone group H5HA and H7HA quite specific ELISA antibody levels. Use of T lymphocyte transformation test, lymphocyte count category, IFN-γsecretion of the number of test data, this results show that the joint contains multiple epitopes that induced immune cells in the immune group optimal level. Proliferation of CD4+ T lymphocytes. H5 and H7 antigen used to stimulate T lymphocyte proliferation and IFN-γsecretion detected. Found that HA, NP components of the immune cells play a major role. Adding epitope improve the specificity of H3 subtype Stimulation Index, that some components have increased epitope immune function.In summary, this study predicted H1, H3 epitope and then design CTL, B/Th epitope expression box. Based on the research front, building a H5, H7 subtypes of influenza virus multi-epitope DNA vaccine. DNA vaccines in inducing cellular immunity and humoral immunity has a significant advantage by immunogenicity. Derived from the H3 subtype of influenza virus epitope-specific cellular immune response has a significant advantage by immunogenicity. In this study, we developed a common influenza vaccine against H1, H3, H5, H7 subtypes of influenza virus and laid the foundation.更多还原