【摘要】： 目的:以減毒沙門氏菌攜帶BPV1-L2(牛乳頭狀瘤病毒1型-L2抗原)表達(dá)質(zhì)粒,滴鼻粘膜免疫小鼠后,觀察小鼠血清及陰道沖洗液中抗HPV16-L1及HPV18-L1抗體產(chǎn)生的情況。以期證實(shí)BPV1-L2單一抗原免疫,可建立對(duì)多種型別人乳頭狀瘤病毒的交叉免疫應(yīng)答。 方法:從牛皮膚贅生物組織基因組DNA中,釣取目的基因BPV1-L2,連接至pMD-18T質(zhì)粒,經(jīng)雙酶切及測(cè)序鑒定正確后,構(gòu)建原核表達(dá)質(zhì)粒pET28a-BPV1-L2,將重組質(zhì)粒轉(zhuǎn)化至感受態(tài)大腸桿菌BL21,IPTG誘導(dǎo)表達(dá)目的蛋白,SDS-PAGE檢測(cè)目的蛋白。以pET28a-BPV1-L2質(zhì)粒為模板,擴(kuò)增BPV1-L2基因,構(gòu)建真核表達(dá)質(zhì)粒pcDNA3.1-BPV1-L2,將重組質(zhì)粒轉(zhuǎn)染至Vero細(xì)胞,SDS-PAGE檢測(cè)目的蛋白。將pcDNA3.1-BPV1-L2質(zhì)粒電轉(zhuǎn)化至減毒沙門氏菌Ty21a和PhoP/PhoQ,于第0、7、21天經(jīng)滴鼻免疫雌性BalB/c小鼠,于第28天收集小鼠血清及陰道沖洗液,應(yīng)用Western bloting、細(xì)胞免疫熒光染色和ELISA方法檢測(cè)血清及陰道沖洗液中抗BPV1-L2抗體、抗HPV16-L1抗體和抗HPV18-L1抗體。ELISA方法檢測(cè)小鼠血清中IL-2和IFN-γ含量。 結(jié)果:成功構(gòu)建原核表達(dá)質(zhì)粒pET28a-BPV1-L2及真核表達(dá)質(zhì)粒pcDNA3.1-BPV1-L2;免疫細(xì)胞熒光染色、Western bloting結(jié)果顯示,免疫血清中含有抗HPV16-L1(人乳頭狀瘤病毒16型L1抗原)的抗體,ELISA檢測(cè)到血清及陰道沖洗物中抗HPV16-L1、HPV18-L1(人乳頭狀瘤病毒18型L1抗原)的抗體,及血清中細(xì)胞因子IL-2、INF-γ,表明以減毒沙門氏菌攜帶BPV1-L2表達(dá)質(zhì)�？杉せ钚∈蟮拿庖邞�(yīng)答,并誘導(dǎo)小鼠產(chǎn)生抗HPV中和性抗體。 結(jié)論:減毒沙門氏菌可作為抗原的運(yùn)載體攜帶其他免疫抗原,經(jīng)粘膜免疫后,能激活機(jī)體對(duì)異源抗原特異性免疫應(yīng)答,其自身還具有CpG佐劑效應(yīng),加強(qiáng)機(jī)體的保護(hù)性免疫應(yīng)答。本實(shí)驗(yàn)證實(shí),以牛乳頭狀瘤病毒L2單一抗原產(chǎn)生的免疫應(yīng)答,可以產(chǎn)生針對(duì)兩個(gè)型別(HPV16及HPV18)人乳頭狀瘤病毒的交叉免疫應(yīng)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).
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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