以减毒沙门氏菌携带BPV1-L2表达质粒诱导HPV中和性抗体的研究
发布时间:2019-06-19 16:19
【摘要】: 目的:以减毒沙门氏菌携带BPV1-L2(牛乳头状瘤病毒1型-L2抗原)表达质粒,滴鼻粘膜免疫小鼠后,观察小鼠血清及阴道冲洗液中抗HPV16-L1及HPV18-L1抗体产生的情况。以期证实BPV1-L2单一抗原免疫,可建立对多种型别人乳头状瘤病毒的交叉免疫应答。 方法:从牛皮肤赘生物组织基因组DNA中,钓取目的基因BPV1-L2,连接至pMD-18T质粒,经双酶切及测序鉴定正确后,构建原核表达质粒pET28a-BPV1-L2,将重组质粒转化至感受态大肠杆菌BL21,IPTG诱导表达目的蛋白,SDS-PAGE检测目的蛋白。以pET28a-BPV1-L2质粒为模板,扩增BPV1-L2基因,构建真核表达质粒pcDNA3.1-BPV1-L2,将重组质粒转染至Vero细胞,SDS-PAGE检测目的蛋白。将pcDNA3.1-BPV1-L2质粒电转化至减毒沙门氏菌Ty21a和PhoP/PhoQ,于第0、7、21天经滴鼻免疫雌性BalB/c小鼠,于第28天收集小鼠血清及阴道冲洗液,应用Western bloting、细胞免疫荧光染色和ELISA方法检测血清及阴道冲洗液中抗BPV1-L2抗体、抗HPV16-L1抗体和抗HPV18-L1抗体。ELISA方法检测小鼠血清中IL-2和IFN-γ含量。 结果:成功构建原核表达质粒pET28a-BPV1-L2及真核表达质粒pcDNA3.1-BPV1-L2;免疫细胞荧光染色、Western bloting结果显示,免疫血清中含有抗HPV16-L1(人乳头状瘤病毒16型L1抗原)的抗体,ELISA检测到血清及阴道冲洗物中抗HPV16-L1、HPV18-L1(人乳头状瘤病毒18型L1抗原)的抗体,及血清中细胞因子IL-2、INF-γ,表明以减毒沙门氏菌携带BPV1-L2表达质粒可激活小鼠的免疫应答,并诱导小鼠产生抗HPV中和性抗体。 结论:减毒沙门氏菌可作为抗原的运载体携带其他免疫抗原,经粘膜免疫后,能激活机体对异源抗原特异性免疫应答,其自身还具有CpG佐剂效应,加强机体的保护性免疫应答。本实验证实,以牛乳头状瘤病毒L2单一抗原产生的免疫应答,可以产生针对两个型别(HPV16及HPV18)人乳头状瘤病毒的交叉免疫应答。
[Abstract]:Objective: To study the anti-HPV16-L1 and HPV18-L1 antibody production in the serum and vaginal douche of mice with attenuated Salmonella carrying BPV1-L2 (bovine papilloma virus type 1-L2 antigen) expression plasmid. In order to confirm the single-antigen immunization of BPV1-L2, the cross-immune response to multiple-type human papilloma virus can be established. Methods: The target gene BPV1-L2 was connected to pMD-18T plasmid from the genomic DNA of the bovine skin neoplasm tissue. The prokaryotic expression plasmid pET28a-BPV1-L2 was constructed by double-enzyme digestion and sequencing. The recombinant plasmid was transformed into the competent E. coli BL21 and the expression was induced by IPTG. Protein, SDS-PAGE for detection of the target And using the pET28a-BPV1-L2 plasmid as a template, amplifying the BPV1-L2 gene, constructing a eukaryotic expression plasmid pcDNA3.1-BPV1-L2, transfecting the recombinant plasmid to Vero cells, and detecting the target by the SDS-PAGE. The pcDNA3.1-BPV1-L2 plasmid was electrically transformed to attenuated Salmonella Ty21a and PhoP/ PhoQ, and the mice were immunized with nasal immune female BalB/ c mice at day 0,7 and 21. The serum and the vaginal douche were collected on the 28th day, and the anti-BPV1-L in the serum and the vaginal rinse was detected by Western blotting, cell immunofluorescence staining and ELISA. 2-antibody, anti-HPV16-L1 antibody and anti-HPV18-L 1. ELISA method for detecting IL-2 and IFN-in mouse serum The results showed that the prokaryotic expression plasmid pET28a-BPV1-L2 and the eukaryotic expression plasmid pcDNA3.1-BPV1-L2 were successfully constructed. The antibodies of PV16-L1, HPV18-L1 (human papillomavirus type 18 L1 antigen), and the serum cytokines IL-2, INF-1, indicated that the expression plasmid carrying the BPV1-L2 by attenuated Salmonella can activate the immune response of the mouse and induce the mouse to produce the anti-H Conclusion: The attenuated Salmonella can be used as the carrier of the antigen to carry other immune antigens, and after the mucosal immunity, the specific immune response of the body to the heterologous antigen can be activated. The protective immune response of the strong body. This experiment confirmed that the immune response produced by the single antigen of the bovine papilloma virus (HPV)2 could result in the papilla of the two types (HPV16 and HPV18).
【学位授予单位】:吉林大学
【学位级别】:硕士
【学位授予年份】:2010
【分类号】:R392
[Abstract]:Objective: To study the anti-HPV16-L1 and HPV18-L1 antibody production in the serum and vaginal douche of mice with attenuated Salmonella carrying BPV1-L2 (bovine papilloma virus type 1-L2 antigen) expression plasmid. In order to confirm the single-antigen immunization of BPV1-L2, the cross-immune response to multiple-type human papilloma virus can be established. Methods: The target gene BPV1-L2 was connected to pMD-18T plasmid from the genomic DNA of the bovine skin neoplasm tissue. The prokaryotic expression plasmid pET28a-BPV1-L2 was constructed by double-enzyme digestion and sequencing. The recombinant plasmid was transformed into the competent E. coli BL21 and the expression was induced by IPTG. Protein, SDS-PAGE for detection of the target And using the pET28a-BPV1-L2 plasmid as a template, amplifying the BPV1-L2 gene, constructing a eukaryotic expression plasmid pcDNA3.1-BPV1-L2, transfecting the recombinant plasmid to Vero cells, and detecting the target by the SDS-PAGE. The pcDNA3.1-BPV1-L2 plasmid was electrically transformed to attenuated Salmonella Ty21a and PhoP/ PhoQ, and the mice were immunized with nasal immune female BalB/ c mice at day 0,7 and 21. The serum and the vaginal douche were collected on the 28th day, and the anti-BPV1-L in the serum and the vaginal rinse was detected by Western blotting, cell immunofluorescence staining and ELISA. 2-antibody, anti-HPV16-L1 antibody and anti-HPV18-L 1. ELISA method for detecting IL-2 and IFN-in mouse serum The results showed that the prokaryotic expression plasmid pET28a-BPV1-L2 and the eukaryotic expression plasmid pcDNA3.1-BPV1-L2 were successfully constructed. The antibodies of PV16-L1, HPV18-L1 (human papillomavirus type 18 L1 antigen), and the serum cytokines IL-2, INF-1, indicated that the expression plasmid carrying the BPV1-L2 by attenuated Salmonella can activate the immune response of the mouse and induce the mouse to produce the anti-H Conclusion: The attenuated Salmonella can be used as the carrier of the antigen to carry other immune antigens, and after the mucosal immunity, the specific immune response of the body to the heterologous antigen can be activated. The protective immune response of the strong body. This experiment confirmed that the immune response produced by the single antigen of the bovine papilloma virus (HPV)2 could result in the papilla of the two types (HPV16 and HPV18).
【学位授予单位】:吉林大学
【学位级别】:硕士
【学位授予年份】:2010
【分类号】:R392
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