本文選題：空腸彎曲桿菌 + HSP43重組蛋自��； 參考：《黑龍江八一農(nóng)墾大學(xué)》2010年碩士論文

【摘要】： 空腸彎曲桿菌(Campylobacter jejuni, C.jejuni)可引起牛冬痢,是一種重要的病原體。本試驗(yàn)以牛源空腸彎曲桿菌及其HSP43重組蛋白(rHSP43)為研究對(duì)象,制備免疫原,對(duì)鼠模型和奶牛進(jìn)行免疫,研究其免疫原性。 根據(jù)GenBank上已發(fā)表的C.jejuni的PorA基因設(shè)計(jì)—對(duì)特異性的引物,應(yīng)用RCR方法擴(kuò)增得到PorA蛋白基因,并克隆到pMD18-T載體上,篩選、鑒定陽性重組子pMD18-T-PorA,然后克隆到原核表達(dá)載體pQE-30a (+)上,經(jīng)雙酶切、PCR鑒定,獲得重組表達(dá)質(zhì)粒pQE-30a-PorA,經(jīng)序列分析證實(shí),構(gòu)建的重組質(zhì)粒pQE-30a-PorA中含有完整的PorA蛋白基因,將含有pQE-30a-PorA的重組菌BL21(DE3),經(jīng)終濃度為1mmol/L的IPTG誘導(dǎo),表達(dá)產(chǎn)物經(jīng)SDS-PAGE分析,結(jié)果顯示C.jejuni-DQ株P(guān)orA蛋白基因在原核表達(dá)系統(tǒng)中獲得高效表達(dá)。Western blot檢測(cè)結(jié)果表明表達(dá)的rHSP43具有良好的反應(yīng)原性。 利用表達(dá)并純化后的rHSP43作為抗原,針對(duì)兩種實(shí)驗(yàn)動(dòng)物,分別建立了檢測(cè)C.jejuni抗體的間接ELISA方法。檢測(cè)小鼠血清抗體的間接ELISA方法的最佳反應(yīng)條件為：抗原的最佳包被液為50mM pH9.6的碳酸鹽緩沖液；抗原包被濃度為7.5μg/mL;封閉液為5%脫脂奶粉,封閉條件為37℃1h；血清稀釋液為5%脫脂奶粉,血清稀釋倍數(shù)為100倍；HRP-羊抗鼠IgG的最適[作濃度為1：5000。檢測(cè)奶牛血清抗體的間接ELISA方法的最佳反應(yīng)條件為：抗原的包被液為50mM pH9.6的碳酸鹽緩沖液；抗原包被濃度為2.5μg/ml；最佳封閉液為5%脫脂奶粉,封閉條件為37℃lh；血清稀釋液為5%脫脂奶粉,血清稀釋倍數(shù)為200倍；HRP-羊抗牛IgG的最適工作濃度為1：5000。包被的重組抗原不與腸炎沙門氏菌、大腸埃希氏桿菌和金黃色葡萄球菌陽性血清發(fā)生交義反應(yīng),表明建立的ELISA方法具有良好的特異性。 本研究采用常規(guī)方法分別制備白油、鋁膠佐劑和不加佐劑的全菌體與rHSP43免疫原,分別以不同劑量經(jīng)皮下、腹腔和灌胃途徑免疫8周齡小鼠,應(yīng)用間接ELISA方法檢測(cè)兩種免疫原免疫小鼠后抗體消長(zhǎng)情況,并通過攻毒試驗(yàn)評(píng)價(jià)其保護(hù)效果。結(jié)果顯示,在全菌體免疫原組中,5x1010CFU/mL組與5×109CFU/mL組產(chǎn)生的特異性抗體水平差異不顯著,二者與5×107CFU/mL組相比,差異顯著(p0.05)；腹腔與皮下注射產(chǎn)生的抗體水平差異不顯著(p0.05),二者明顯高于灌胃途徑(P0.05)；白油佐劑組產(chǎn)生的抗體水平要高于鋁膠佐劑組,二者明顯高于未加佐劑組(P0.05)。rHSP43免疫原組中,免疫途徑的效果比較和佐劑的效果比較結(jié)果與全菌體免疫原相似。在小鼠免疫后21 d以1×1010劑量的C.jejuni攻菌,以白油佐劑乳化的全菌體免疫原皮下注射能夠提供完全保護(hù),而rHSP43僅能提供75%的保護(hù)。將白油佐劑乳化的全菌體免疫原通過頸部皮下注射免疫奶牛,免疫后血清中產(chǎn)生了高水平的C.jejuni特異性IgG,與對(duì)照組差異極顯著(p0.01)。小鼠和青年牛免疫試驗(yàn)表明,全菌體與rHSP43免疫原均能夠誘導(dǎo)機(jī)體產(chǎn)生特異性抗體,小鼠攻毒試驗(yàn)證明兩種免疫原均能有效預(yù)防C.jejuni攻擊,但全菌體免疫原效果更好。
[Abstract]:Campylobacter jejuni (C.jejuni) is an important pathogen of bovine bacilli. In this experiment, the immunogenicity of Campylobacter jejuni and its HSP43 recombinant protein (rHSP43) was studied. Immunogen was prepared. The immunogenicity of rat model and cow was immunized and its immunogenicity was studied.
According to the PorA gene design of C.jejuni published on GenBank - specific primers, the PorA protein gene was amplified by RCR method and was cloned to pMD18-T vector. The positive recombinant pMD18-T-PorA was screened and identified and then cloned to the prokaryotic expression vector pQE-30a (+). The recombinant expression plasmid pQE-30a-Po was obtained by double enzyme cutting and PCR identification. The recombinant expression plasmid pQE-30a-Po was obtained. RA, sequence analysis confirmed that the recombinant plasmid pQE-30a-PorA contained a complete PorA protein gene, which contained the recombinant strain BL21 (DE3) containing pQE-30a-PorA, induced by IPTG of the final concentration of 1mmol/L, and the expression product was analyzed by SDS-PAGE. The results showed that the PorA protein gene of C.jejuni-DQ strain was highly expressed in the prokaryotic expression system. The results showed that the expressed rHSP43 had good reactivity.
Using the expressed and purified rHSP43 as an antigen, the indirect ELISA method for detecting C.jejuni antibody was established for two experimental animals. The best reaction condition for the indirect ELISA method for detecting the serum antibody of mice was that the best envelope of the antigen was 50mM pH9.6, the concentration of the antigen was 7.5 mu g/mL; For 5% skim milk powder, the closed condition was 37 1H, the serum dilution was 5% skimmed milk powder and the serum dilution multiplier was 100 times. The optimum reaction condition for the indirect ELISA method of HRP- Sheep anti mouse IgG was as follows: the antigen inclusion fluid was the carbonate buffer solution of 50mM pH9.6; the antigen inclusion concentration was the concentration. The best closed liquid was 5% skimmed milk powder, the closed condition was 37 LH, and the serum diluent was 5% skimmed milk powder, and the serum dilution multiple was 200 times; the optimum working concentration of HRP- Sheep anti bovine IgG for 1:5000. package was not with Salmonella enteritis, Escherichia coli and Staphylococcus aureus positive serum. The results showed that the established ELISA method had good specificity.
In this study, white oil, aluminum gum adjuvant and non adjuvant all bacteria and rHSP43 immunogen were prepared by routine methods. 8 weeks old mice were immunized with different doses of subcutaneous, peritoneal and intraperitoneal methods respectively. Indirect ELISA method was used to detect the antibody growth of two immunogenic mice, and the protective effect was evaluated by attack test. The results showed that there was no significant difference in the level of specific antibody produced by the group 5x1010CFU/mL and the 5 x 109CFU/mL group in the whole bacterial immunogen group. The difference was significant (P0.05) compared with the 5 x 107CFU/mL group, and the difference in the level of antibody produced by the intraperitoneal and subcutaneous injection was not significant (P0.05), and the two were significantly higher than the intragastric pathway (P0.05); the white oil adjuvant group was produced. The level of antibody was higher than that of the aluminum gel adjuvant group. The two was significantly higher than that in the.RHSP43 immunogen group without the adjuvant group (P0.05). The results of the immunization pathway and the effect of the adjuvant were similar to those of the whole bacteria immunogen. The 21 d after the immunization of the mice was 1 * 1010 doses of C.jejuni, and the whole bacterial immunogen emulsified with the white oil adjuvant was injected subcutaneously. Complete protection can be provided, and rHSP43 can provide only 75% protection. The whole bacterial immunogen of the emulsified oil adjuvant is immunized by the subcutaneous injection of the cow. The high level of C.jejuni specific IgG is produced in the serum after immunization. The difference is very significant (P0.01) with the control group (P0.01). The mice and the green year bovine immune test showed that the whole fungus and the rHSP43 immunogen Both of them could induce specific antibody production in the body. The attack test showed that all two immunogens could effectively prevent C.jejuni attacks, but the whole cell immunogen was better.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐懷英,秦卓明,何葉峰,歐陽文軍,王傳強(qiáng),金維江;不同型號(hào)白油ND油乳劑滅活苗免疫比較實(shí)驗(yàn)[J];山東家禽;2002年10期

2 李廣興,劉建平,周志勇;雞空腸彎曲菌滅活苗的制備及其免疫保護(hù)試驗(yàn)研究[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2003年03期

3 侯衛(wèi)東;袁遠(yuǎn);張?chǎng)?;牛病毒性腹瀉囊素油乳劑滅活疫苗的研制[J];河南畜牧獸醫(yī);2006年03期

4 張旭輝;熱休克蛋白70與熱耐受的機(jī)制[J];解放軍預(yù)防醫(yī)學(xué)雜志;2001年01期

5 李成文;王栩;高燕;孫萬邦;余妍;馮勝軍;;空腸彎曲菌外膜蛋白亞單位疫苗的研制及免疫原性研究[J];免疫學(xué)雜志;2008年04期

6 黃曉兵;熱休克蛋白70與腫瘤細(xì)胞凋亡[J];四川腫瘤防治;2003年01期

7 高建新,王煥妞,謝根甫;甲醛化空腸彎曲菌菌苗誘導(dǎo)腸道免疫應(yīng)答機(jī)理的初步研究[J];上海免疫學(xué)雜志;1988年03期

8 高建新 ,謝雅莉 ,馬寶驪;空彎菌苗誘導(dǎo)不同品系小鼠的腸道粘膜免疫應(yīng)答[J];上海免疫學(xué)雜志;1990年02期

9 郭津生,王吉耀,紀(jì)元,譚云山;熱休克蛋白47在人肝癌癌周組織和大鼠胃潰瘍組織中的表達(dá)和意義[J];胃腸病學(xué)和肝病學(xué)雜志;2000年04期

10 馮勝軍,孫萬邦,姚新生,肖政;空腸彎曲菌28-31 ku外膜蛋白的免疫原性[J];世界華人消化雜志;2004年12期

，

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