本文關(guān)鍵詞：淋球菌IgA蛋白酶中和表位表達(dá)載體的構(gòu)建及其誘導(dǎo)小鼠的粘膜免疫　出處：《南華大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

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【摘要】： 【目的】: (1)構(gòu)建淋球菌IgA蛋白酶中和表位原核表達(dá)載體,并在大腸桿菌中誘導(dǎo)表達(dá),純化表達(dá)產(chǎn)物6His-IgA蛋白酶中和表位融合蛋白,檢測融合蛋白的免疫原性。 (2)構(gòu)建淋球菌IgA蛋白酶中和表位真核表達(dá)載體,通過生殖道粘膜途徑接種,在動(dòng)物體內(nèi)表達(dá)相應(yīng)產(chǎn)物,誘導(dǎo)粘膜免疫,為研制人用高效抗淋病核酸疫苗提供實(shí)驗(yàn)依據(jù)。 【方法】: (1)IgA蛋白酶中和表位原核表達(dá)載體的構(gòu)建、表達(dá)、純化及其免疫原性鑒定通過PCR擴(kuò)增淋球菌MS11株IgA蛋白酶編碼基因1 601～2 722位序列,構(gòu)建pQE30-IgA蛋白酶中和表位原核表達(dá)載體;測序正確后在大腸桿菌M15中誘導(dǎo)表達(dá),包涵體重組蛋白經(jīng)8M尿素變性后通過鎳瓊脂凝膠FF分離純化,SDS-PAGE和Western-Blot分析及鑒定該純化蛋白;后者經(jīng)尿素濃度梯度透析復(fù)性、SDS-PAGE鑒定正確后,與佐劑混合于皮內(nèi)多點(diǎn)注射免疫新西蘭兔,誘導(dǎo)產(chǎn)生抗淋球菌IgA蛋白酶中和表位的多克隆抗體;以復(fù)性后的重組蛋白抗原包板,建立間接ELISA法,檢測兔免疫血清中IgA蛋白酶中和表位的抗體效價(jià)。 (2)構(gòu)建IgA蛋白酶中和表位真核表達(dá)載體,誘導(dǎo)小鼠產(chǎn)生粘膜免疫通過PCR擴(kuò)增淋球菌MS11株IgA蛋白酶編碼基因1 601-2 722位序列,構(gòu)建pcDNA3.1(-)-IgA蛋白酶中和表位真核表達(dá)載體;經(jīng)鑒定后,制備重組質(zhì)粒殼聚糖混合顆粒,通過陰道灌注法免疫小鼠,誘導(dǎo)其產(chǎn)生粘膜免疫,對照組免疫不含重組質(zhì)粒的殼聚糖溶液和pcDNA3.1(-)殼聚糖混合顆粒溶液;第3周和第5周加強(qiáng)免疫一次,間接免疫熒光法檢測目的抗原在小鼠陰道組織內(nèi)的表達(dá),ELISA法檢測小鼠血清陰道沖洗液中抗IgA蛋白酶中和表位的IgA和IgG類抗體。 【結(jié)果】: (1)成功構(gòu)建了淋球菌IgA蛋白酶中和表位的原核表達(dá)載體。通過PCR擴(kuò)增獲得的淋球菌IgA蛋白酶中和表位序列與GenBank報(bào)道的一致。所克隆的基因在大腸桿菌中獲得高效表達(dá),目的蛋白在菌體細(xì)胞內(nèi)以包涵體形式存在,Western-Blot結(jié)果顯示該純化蛋白能被抗6-His單抗識(shí)別,復(fù)性后得到溶解狀態(tài)的復(fù)性蛋白。免疫組兔血清中IgA蛋白酶中和表位抗體效價(jià)達(dá)1:6 400。 (2)成功構(gòu)建了淋球菌IgA蛋白酶中和表位真核表達(dá)載體。以殼聚糖為釋放系統(tǒng)經(jīng)陰道免疫后第4天開始,通過間接免疫熒光法檢測,在免疫組小鼠陰道上皮細(xì)胞中可以觀察到一定亮度的綠色熒光,提示IgA蛋白酶中和表位抗原能在小鼠陰道組織內(nèi)表達(dá)。免疫組小鼠陰道沖洗液中檢測到的抗IgA蛋白酶中和表位的sIgA水平明顯高于對照組。 【結(jié)論】: (1)成功構(gòu)建了淋球菌pQE30-IgA蛋白酶中和表位原核表達(dá)載體,免疫動(dòng)物后獲得了具有較好免疫原性的純化復(fù)性IgA蛋白酶中和表位蛋白。 (2)成功構(gòu)建了淋球菌pcDNA3.1(-)-IgA蛋白酶中和表位真核表達(dá)載體,其殼聚糖混合顆粒經(jīng)陰道免疫小鼠,能誘導(dǎo)產(chǎn)生有效的生殖道粘膜免疫。 (3)pcDNA3.1(-)-IgA蛋白酶中和表位蛋白能在小鼠陰道上皮組織內(nèi)表達(dá)。
[Abstract]:[Objective]:
(1) construct prokaryotic expression vector of Neisseria gonorrhoeae IgA protease neutralization epitope, and express it in Escherichia coli, and purify 6His-IgA protein and neutralization epitope fusion protein, and detect the immunogenicity of fusion protein.
(2) we constructed the eukaryotic expression vector of Neisseria gonorrhoeae IgA protease neutralization epitope, and expressed the corresponding products in the animal body through the inoculation of genital tract mucosal channel, and induced mucosal immunity, so as to provide experimental evidence for developing highly effective anti gonorrhea nucleic acid vaccine.
[method]:
(1) IgA neutralizing epitope prokaryotic expression vector construction, expression, purification and immunogenicity identification of Neisseria gonorrhoeae strain MS11 IgA protease encoding gene was amplified by PCR from 1601 to 2722 sequences, construct pQE30-IgA neutralizing epitope prokaryotic expression vector; after sequencing in Escherichia coli M15, inclusion body group of protein by nickel agar gel FF purification by 8M urea denaturation, analysis and identification of SDS-PAGE and Western-Blot of the purified protein; the latter by urea gradient dialysis refolding, SDS-PAGE after correct identification, with the adjuvants in New Zealand multi point injection induced immune rabbit polyclonal antibody against gonococcal IgA protease neutralizing epitope; package board with recombinant protein antigen after refolding, an indirect ELISA method, detection of rabbit immune serum neutralization antibody titer of a IgA protease.
(2) to construct IgA neutralizing epitope of eukaryotic expression vector of mice induced by mucosal immune IgA protease encoding gene of Neisseria gonorrhoeae strain MS11 1 601-2 722 bit sequence was amplified by PCR to construct pcDNA3.1 (-) -IgA neutralizing epitope of eukaryotic expression vector; after identification, preparation of recombinant plasmid chitosan mixed particles. Through the vagina perfusion in mice to induce mucosal immunity, the immune control group without recombinant plasmid chitosan solution and pcDNA3.1 (-) chitosan mixed particle solution; a second immunization for third weeks and fifth weeks, to detect the expression of target antigen indirect immunofluorescence method in mouse vaginal tissue, serum ELISA? Vaginal washing liquid and anti IgA protease IgA and IgG antibody level.
[results]:
(1) construct a prokaryotic expression vector of gonococcal IgA protease neutralizing epitope. Gonococcal IgA protease amplified by PCR neutralizing epitope sequence reported in GenBank. The cloned gene was expressed in Escherichia coli, the protein in the cell in the form of inclusion bodies, the results of Western-Blot the purified protein can be anti 6-His monoclonal antibody recognition, renaturation after refolding protein dissolved state. IgA protease immunohistochemistry in rabbit serum neutralizing epitope antibody titer was 1:6 400.
(2) the successful construction of gonococcal IgA protease neutralizing epitope of eukaryotic expression vector. Using chitosan as vaginal delivery system fourth days after immunization, by indirect immunofluorescence assay, in immunized mice vaginal epithelial cells can be observed in certain brightness green fluorescence, suggesting that IgA neutralizing epitope the expression in murine vaginal tissue. Immunized mice vaginal washing fluid was detected in the anti IgA neutralizing epitope of sIgA was significantly higher than control group.
[Conclusion]:
(1) the prokaryotic expression vector of Neisseria gonorrhoeae pQE30-IgA protease neutralization epitope was successfully constructed. After immunization, the neutralizing epitope protein of purified IgA protease was obtained.
(2) we successfully constructed the eukaryotic expression vector of pcDNA3.1 (-) -IgA protease neutralization epitope, and its chitosan mixed particles could induce effective mucosal immunity in mice through immunization with vagina.
(3) the pcDNA3.1 (-) -IgA protease neutralization epitope protein can be expressed in the mouse vaginal epithelial tissue.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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本文編號(hào)：1413551