霍乱DNA疫苗和灭活疫苗联合免疫诱导系统和肠道粘膜抗体应答的研究

发布时间：2018-04-25 06:27

本文选题：霍乱弧菌 + 霍乱疫苗　；参考：《南京医科大学》2009年博士论文

【摘要】：第一部分密码子优化的霍乱肠毒素B亚单位DNA疫苗的免疫原性及保护作用研究目的:构建密码子优化的霍乱肠毒素B亚单位(CTB)DNA疫苗,并研究其免疫原性以及诱导的保护性免疫应答。方法:根据CTB氨基酸序列资料,按哺乳动物细胞和大肠杆菌偏好的密码子对CTB基因序列进行密码子优化合成优化基因,优化的基因既可用于在哺乳动物细胞表达,也可在大肠杆菌中的表达。然后,将密码子优化的CTB基因克隆到核酸疫苗载体pJW4303,构建CTB DNA疫苗。用CTB DNA疫苗转染293T细胞,通过蛋白质免疫印迹法检测转染细胞中CTB蛋白的表达。在此基础上,采用肌肉注射+活体基因导入方式免疫,用CTB DNA疫苗免疫新西兰兔和Balb/c小鼠。兔免疫的时间点为第0、2、4和8周,而小鼠于第0和2周进行免疫。于免疫前和每次免疫后两周收集血清和粪便标本,通过ELISA方法检测免疫后小鼠和兔血清及粪便中CTB特异性IgG抗体应答。用乳鼠保护试验检测免疫兔血清对致病性O1群霍乱弧菌Eltor生物型菌株的的被动保护作用,免疫兔肠段结扎试验验证CTB DNA疫苗免疫产生的主动保护作用。结果:构建的密码子优化的CTB DNA疫苗,能正确表达CTB蛋白。CTB DNA疫苗免疫家兔和小鼠后,不仅可以血清中能产生较高水平的CTB特异性的抗体,而且粪便中也能检测到CTB特异性IgG抗体应答。乳鼠保护试验和兔肠段结扎试验表明,CTB DNA疫苗免疫后,可以诱导机体产生保护性免疫反应。结论:密码子优化的CTB DNA疫苗具有良好的免疫原性,在动物体内能诱导产生保护性免疫应答。第二部分CTB DNA疫苗初免及KWC-B疫苗胃肠道外加强诱导系统性和粘膜抗体应答的研究目的:研究霍乱CTB DNA疫苗初免和灭活全菌体/霍乱肠毒素B亚单位(KWC-B)疫苗胃肠道外加强联合免疫诱导的系统性和粘膜抗体应答。方法:Balb/c小鼠于第0和2周免疫两次,分别接受同源的DNA初免+DNA加强(DNA+DNA),KWC-B初免+ KWC-B加强(KWC-B+KWC-B),或者异源性DNA初免+ KWC-B加强(DNA+KWC-B)。DNA疫苗通过肌肉注射、活体基因导入免疫,KWC-B肌肉注射。免疫前和免疫后2周采血和收集小鼠粪便。ELISA方法检测免疫血清和粪便中CTB特异性IgG、IgA和IgM抗体应答以及血清中CTB特异性IgG抗体亚型。第二次免疫后5个月处死小鼠,分离脾细胞,通过ELISPOT检测CTB特异性IgG抗体分泌细胞。结果:KWC-B疫苗一次肌肉注射免疫不能诱导血清高水平IgG、IgA和IgM抗体应答,粪便中不能检测到抗体应答。两次免疫后,小鼠接受CTB DNA疫苗单独免疫,KWC-B疫苗单独免疫,或CTB DNA疫苗初免及KWC-B疫苗加强组血清中均产生了高水平的抗体应答,粪便中也检测到IgG。KWC-B单独免疫诱导以IgG1为主,Th2型抗体应答,而DNA疫苗单独免疫诱导较好IgG2a,Th1型抗体应答。有趣的是,CTB DNA疫苗初免出现更平衡的IgG1和IgG2a,更加平衡的Th1/Th2应答。所有接受两次免疫的组别检测到CTB特异性抗体分泌细胞。结论:CTB DNA疫苗单独免疫,KWC-B(IM)单独免疫,或CTB DNA疫苗初免-KWC-B疫苗(IM)强联合免疫组小鼠均诱导抗CTB的系统性和粘膜抗体应答。然而,CTB DNA疫苗初免-KWC-B疫苗(IM)加强诱导更加平衡的Th1/Th2抗体应答。第三部分CTB DNA疫苗初免及KWC-B疫苗口服加强诱导系统性和粘膜抗体应答的研究目的:研究霍乱CTB DNA疫苗初免及KWC-B疫苗口服加强联合免疫诱导的系统性和粘膜抗体应答。方法: Balb/c小鼠于第0和2周免疫,分别接受同源的DNA初免+DNA加强(DNA+DNA),KWC-B初免+ KWC-B加强(KWC-B(O)+KWC-B(O)),或者异源性DNA初免+ KWC-B加强(DNA+KWC-B(O))。DNA疫苗通过肌肉注射、活体基因导入免疫,KWC-B疫苗经口免疫。免疫前和免疫后2周采血和收集小鼠粪便。ELISA方法检测免疫血清和粪便中CTB特异性IgG、IgA和IgM抗体应答以及血清中CTB特异性IgG抗体亚型。第二次免疫后5个月处死小鼠,分离脾细胞,通过ELISPOT检测CTB特异性IgG抗体分泌细胞。结果:KWC-B疫苗口服一次免疫未能诱导血清和粪便特异性抗体应答。两次免疫后, CTB DNA疫苗单独免疫组,或CTB DNA疫苗初免及KWC-B疫苗口服加强组小鼠血清中产生了高水平的抗体应答(IgG、IgA和IgM)。KWC-B疫苗单独口服组粪便中仅检测到IgA,而DNA疫苗单独免疫组仅检测到IgG。有趣的是,接受CTB DNA疫苗初免及KWC-B口服加强组粪便中检测到IgA和IgG应答。第二次免疫后5个月,CTB特异性抗体分泌细胞仅在CTB DNA疫苗单独免疫组,及CTB DNA初免+KWC-B口服加强组检测到,KWC-B单独免疫组未检测到。结论:虽然KWC-B口服免疫诱导粘膜抗体的应答,但是不能诱导系统性抗体应答。与DNA单独免疫或KWC-B单独免疫相比,CTB DNA初免+KWC-B口服加强诱导更强的系统性和粘膜抗体应答。
[Abstract]:Part one: immunogenicity and protection of cholera enterotoxin B subunit DNA vaccine optimized by codon optimization
Objective: to construct a codon optimized cholera enterotoxin B subunit (CTB) DNA vaccine, and to study its immunogenicity and induced protective immune response.
Methods: according to the CTB amino acid sequence data, the optimal genes were optimized by the codon of mammalian and Escherichia coli to optimize the CTB gene sequence. The optimized gene could be used in both mammalian and Escherichia coli, and then the CTB gene was cloned to the nucleic acid vaccine. Carrier pJW4303, CTB DNA vaccine was constructed. 293T cells were transfected with CTB DNA vaccine and the expression of CTB protein in transfected cells was detected by protein immunoblotting. On this basis, immunization with intramuscular injection + living gene introduction was used and CTB DNA vaccine was used to immunize New Zealand rabbits and Balb/c mice. The time point of immunization of rabbits was 0,2,4 and 8 weeks, and small The mice were immunized at zeroth and second weeks. The serum and fecal specimens were collected before and two weeks after immunization. The specific IgG antibody response to CTB in serum and feces of the mice and rabbits after immunization was detected by ELISA method. The passive protective effect of the serum of the immune rabbit on the Eltor biotype of the pathogenic O1 group of the cholera cholera was detected by the protection test of the milk rat. The rabbit intestinal segment ligation test confirmed the active protective effect of CTB DNA vaccine immunization.
Results: the constructed codon optimized CTB DNA vaccine can correctly express the CTB protein.CTB DNA vaccine to immunize rabbits and mice, not only can produce a high level of CTB specific antibodies in the serum, but also detect the CTB specific IgG antibody response in the feces. The milk rat protection test and the rabbit intestinal segment ligation test show CTB DNA vaccine. After immunization, a protective immune response can be induced.
Conclusion: the codon optimized CTB DNA vaccine has good immunogenicity and can induce a protective immune response in animals.
The second part is the study of systemic immunity and mucosal antibody response induced by CTB DNA vaccine and KWC-B vaccine.
Objective: To study the systemic and mucosal antibody response induced by combined immunization of CTB DNA vaccine against cholera and inactivated total bacteria / cholera enterotoxin B subunit (KWC-B) vaccine.
Methods: Balb/c mice were immunized two times in zeroth and second weeks, receiving the homologous DNA primer free +DNA strengthening (DNA+DNA), KWC-B primer + KWC-B strengthening (KWC-B+KWC-B), or heterologous DNA primer + KWC-B strengthening (DNA+KWC-B).DNA vaccine by intramuscular injection. The living gene was introduced into the immune system, the KWC-B muscles were injected. The blood was collected and collected at 2 weeks before and after immunization. The rat fecal.ELISA method was used to detect CTB specific IgG, IgA and IgM antibody response and the CTB specific IgG antibody subtype in serum. The mice were killed, splenic cells were isolated, and the CTB specific IgG antibody secreting cells were detected by ELISPOT for 5 months after second immunizations. At the high level of IgG, IgA and IgM antibody responses, the antibody response could not be detected in the feces. After two immunizations, the mice were immunized by the CTB DNA vaccine alone, the KWC-B vaccine was immunized alone, or the sera of the CTB DNA vaccine primer and the KWC-B vaccine reinforced group all produced a high level of antibody response, and the excrement was also detected by IgG.KWC-B alone. Main, Th2 type antibody response, and DNA vaccine alone immunized to induce better IgG2a, Th1 type antibody response. Interestingly, the CTB DNA vaccine first exempted a more balanced IgG1 and IgG2a, and a more balanced Th1/Th2 response. All two immunization groups detected CTB specific antibody secreting cells.
Conclusion: CTB DNA vaccine is immunized alone, KWC-B (IM) is individually immune, or CTB DNA vaccine primer free -KWC-B vaccine (IM) strong joint immune group can induce systemic and mucosal antibody response to CTB. However, CTB DNA vaccine primer vaccine enhances a more balanced antibody response.
The third part is CTB DNA vaccination and KWC-B vaccine oral induction to enhance systemic and mucosal antibody responses.
Objective: To study the systemic and mucosal antibody responses induced by initial immunization of cholera CTB DNA vaccine and oral immunization with KWC-B vaccine.
Methods: Balb/c mice were immunized for zeroth and second weeks, receiving homologous DNA primordial +DNA strengthening (DNA+DNA), KWC-B primordial immunization + KWC-B strengthening (KWC-B (O) +KWC-B (O)), or heterologous DNA primordial KWC-B enhancement (DNA+DNA) vaccine by intramuscular injection, immunization of the living body gene, immunization of the vaccine through the mouth, and blood sampling before and after immunization. The mouse feces.ELISA method was collected to detect the CTB specific IgG, IgA and IgM antibody responses and the CTB specific IgG antibody subtypes in the serum. The mice were killed 5 months after second immunizations, splenic cells were isolated, and CTB specific IgG antibody secreting cells were detected by ELISPOT.
Results: KWC-B vaccine failed to induce serum and fecal specific antibody response. After two times of immunization, CTB DNA vaccine alone, or CTB DNA vaccine primordial immunity and KWC-B vaccine oral strengthening group produced a high level of antibody response (IgG, IgA and IgM).KWC-B vaccine alone in the faeces of the single oral group detected only IgA, The DNA vaccine alone detected only IgG., which was interesting to detect IgA and IgG responses in the faeces of CTB DNA vaccine primordial exemptions and KWC-B oral strengthening groups. 5 months after the second immunization, CTB specific antibody secreting cells were detected only in the CTB DNA vaccine alone and in the CTB DNA primordial plus oral strengthening group. It was detected.
Conclusion: Although KWC-B immunization induced the response of mucosal antibody, it does not induce systemic antibody response. Compared with DNA alone or KWC-B alone, CTB DNA primordial +KWC-B oral administration strengthens the stronger systemic and mucosal antibody response.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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