Sao蛋白多克隆抗體制備鑒定及促全血?dú)⒕饔?/H1>
發(fā)布時間：2018-07-12 16:51
本文選題：型豬鏈球菌 + 原核表達(dá)��； 參考：《中國公共衛(wèi)生》2014年03期
【摘要】：目的制備2型豬鏈球菌(S.suis 2)表面抗原一(Sao)重組蛋白的多克隆抗體,鑒定其免疫學(xué)特性。方法通過PCR從2型豬鏈球菌中國強(qiáng)毒株05ZYH33基因組中擴(kuò)增基因Sao,構(gòu)建重組表達(dá)質(zhì)粒pET28a-Sao,轉(zhuǎn)化E.coli BL21,異丙基硫代半乳糖苷誘導(dǎo)表達(dá)目的蛋白,十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳鑒定表達(dá)產(chǎn)物,His親和層析柱純化重組蛋白,利用純化后Sao蛋白制備多克隆抗體,間接酶聯(lián)免疫吸附試驗檢測抗體效價,Western blot鑒定抗體特異性,評價Sao多克隆抗體的促全血?dú)⒕饔谩＝Y(jié)果構(gòu)建的重組質(zhì)�？梢栽谒拗骶懈咝П磉_(dá),純化后獲得的重組蛋白純度可達(dá)90%;間接酶聯(lián)免疫吸附試驗測定Sao多克隆抗體效價為1∶102400;Western blot結(jié)果顯示,Sao多克隆抗體有較好的特異性;全血?dú)⒕鷮嶒烇@示,05ZYH33在含有Sao多抗血清的全血中相對存活率下降85%。結(jié)論本研究制備的2型豬鏈球菌重組Sao蛋白多克隆抗體效價高,特異性好,可明顯增強(qiáng)全血對細(xì)菌的殺傷作用。
[Abstract]:Objective to prepare the polyclonal antibody against the surface antigen 1 (Sao) recombinant protein of S.suis 2 and to identify its immunological properties. Methods the gene Sao. was amplified from the genome of strain 05ZYH33 of Streptococcus suis by PCR. The recombinant expression plasmid pET28a-Saowas constructed and transformed into E. coli BL21, and the target protein was induced by isopropylthiogalactoside. Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis was used to identify the recombinant protein by his affinity chromatography. The polyclonal antibody was prepared by using the purified Sao protein. Indirect enzyme-linked immunosorbent assay (Elisa) was used to detect the titer of antibodies. Western blot was used to identify the specificity of antibodies and to evaluate the bactericidal effect of Sao polyclonal antibodies on whole blood. Results the recombinant plasmid could be highly expressed in the host bacteria. The purity of the purified recombinant protein was 90%, and the titer of Sao polyclonal antibody was 1: 102400 by indirect enzyme-linked immunosorbent assay (Elisa). The results showed that the Sao polyclonal antibody had good specificity. Whole blood bactericidal test showed that the relative survival rate of ZYH33 in whole blood containing Sao polyantibody decreased 85%. Conclusion the recombinant Sao protein polyclonal antibody prepared in this study has high titer and good specificity, and can obviously enhance the killing effect of whole blood on bacteria.
【作者單位】： 溫州醫(yī)科大學(xué)檢驗醫(yī)學(xué)院生命科學(xué)學(xué)院;南京軍區(qū)軍事醫(yī)學(xué)研究所;
【基金】：國家自然科學(xué)基金(81172794;81071317;31170124) 江蘇省自然科學(xué)基金(BK2011098;BK2011097) 浙江省新苗人才計劃(2013R413039) 南京軍區(qū)醫(yī)學(xué)科技創(chuàng)新課題(ZX39)
【分類號】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 邵珠卿;韓明月;葛俊超;王長軍;潘秀珍;唐家琪;;豬鏈球菌2型組氨酸三聚體蛋白原核表達(dá)[J];中國公共衛(wèi)生;2009年06期
【共引文獻(xiàn)】
相關(guān)期刊論文 前2條
1 邵珠卿;潘秀珍;韓明月;李先富;秦躍紅;王長軍;唐家琪;;組氨酸三聚體蛋白在2型豬鏈球菌的定位[J];臨床檢驗雜志;2009年06期
2 侯田青;郭靜靜;邵珠卿;李先富;王長軍;潘秀珍;;2型豬鏈球菌組氨酸三聚體蛋白家族05SSU1267的克隆、表達(dá)及免疫保護(hù)作用研究[J];免疫學(xué)雜志;2012年03期
相關(guān)博士學(xué)位論文 前1條
1 孫學(xué)強(qiáng);馬皰疹病毒1型感染性克隆及表達(dá)豬鏈球菌2型亞單位重組病毒的構(gòu)建[D];南京農(nóng)業(yè)大學(xué);2010年
【二級參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 呼顯生;彭召玲;李靜姬;馬玉敏;姜成;;1株豬源性鏈球菌的分離鑒定及藥敏試驗[J];中國公共衛(wèi)生;2006年10期
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
1 吳曉e，
本文編號：2117808
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2117808.html
本文選題：型豬鏈球菌 + 原核表達(dá)��； 參考：《中國公共衛(wèi)生》2014年03期
【摘要】：目的制備2型豬鏈球菌(S.suis 2)表面抗原一(Sao)重組蛋白的多克隆抗體,鑒定其免疫學(xué)特性。方法通過PCR從2型豬鏈球菌中國強(qiáng)毒株05ZYH33基因組中擴(kuò)增基因Sao,構(gòu)建重組表達(dá)質(zhì)粒pET28a-Sao,轉(zhuǎn)化E.coli BL21,異丙基硫代半乳糖苷誘導(dǎo)表達(dá)目的蛋白,十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳鑒定表達(dá)產(chǎn)物,His親和層析柱純化重組蛋白,利用純化后Sao蛋白制備多克隆抗體,間接酶聯(lián)免疫吸附試驗檢測抗體效價,Western blot鑒定抗體特異性,評價Sao多克隆抗體的促全血?dú)⒕饔谩＝Y(jié)果構(gòu)建的重組質(zhì)�？梢栽谒拗骶懈咝П磉_(dá),純化后獲得的重組蛋白純度可達(dá)90%;間接酶聯(lián)免疫吸附試驗測定Sao多克隆抗體效價為1∶102400;Western blot結(jié)果顯示,Sao多克隆抗體有較好的特異性;全血?dú)⒕鷮嶒烇@示,05ZYH33在含有Sao多抗血清的全血中相對存活率下降85%。結(jié)論本研究制備的2型豬鏈球菌重組Sao蛋白多克隆抗體效價高,特異性好,可明顯增強(qiáng)全血對細(xì)菌的殺傷作用。
[Abstract]:Objective to prepare the polyclonal antibody against the surface antigen 1 (Sao) recombinant protein of S.suis 2 and to identify its immunological properties. Methods the gene Sao. was amplified from the genome of strain 05ZYH33 of Streptococcus suis by PCR. The recombinant expression plasmid pET28a-Saowas constructed and transformed into E. coli BL21, and the target protein was induced by isopropylthiogalactoside. Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis was used to identify the recombinant protein by his affinity chromatography. The polyclonal antibody was prepared by using the purified Sao protein. Indirect enzyme-linked immunosorbent assay (Elisa) was used to detect the titer of antibodies. Western blot was used to identify the specificity of antibodies and to evaluate the bactericidal effect of Sao polyclonal antibodies on whole blood. Results the recombinant plasmid could be highly expressed in the host bacteria. The purity of the purified recombinant protein was 90%, and the titer of Sao polyclonal antibody was 1: 102400 by indirect enzyme-linked immunosorbent assay (Elisa). The results showed that the Sao polyclonal antibody had good specificity. Whole blood bactericidal test showed that the relative survival rate of ZYH33 in whole blood containing Sao polyantibody decreased 85%. Conclusion the recombinant Sao protein polyclonal antibody prepared in this study has high titer and good specificity, and can obviously enhance the killing effect of whole blood on bacteria.
【作者單位】： 溫州醫(yī)科大學(xué)檢驗醫(yī)學(xué)院生命科學(xué)學(xué)院;南京軍區(qū)軍事醫(yī)學(xué)研究所;
【基金】：國家自然科學(xué)基金(81172794;81071317;31170124) 江蘇省自然科學(xué)基金(BK2011098;BK2011097) 浙江省新苗人才計劃(2013R413039) 南京軍區(qū)醫(yī)學(xué)科技創(chuàng)新課題(ZX39)
【分類號】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 邵珠卿;韓明月;葛俊超;王長軍;潘秀珍;唐家琪;;豬鏈球菌2型組氨酸三聚體蛋白原核表達(dá)[J];中國公共衛(wèi)生;2009年06期
【共引文獻(xiàn)】
相關(guān)期刊論文 前2條
1 邵珠卿;潘秀珍;韓明月;李先富;秦躍紅;王長軍;唐家琪;;組氨酸三聚體蛋白在2型豬鏈球菌的定位[J];臨床檢驗雜志;2009年06期
2 侯田青;郭靜靜;邵珠卿;李先富;王長軍;潘秀珍;;2型豬鏈球菌組氨酸三聚體蛋白家族05SSU1267的克隆、表達(dá)及免疫保護(hù)作用研究[J];免疫學(xué)雜志;2012年03期
相關(guān)博士學(xué)位論文 前1條
1 孫學(xué)強(qiáng);馬皰疹病毒1型感染性克隆及表達(dá)豬鏈球菌2型亞單位重組病毒的構(gòu)建[D];南京農(nóng)業(yè)大學(xué);2010年
【二級參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 呼顯生;彭召玲;李靜姬;馬玉敏;姜成;;1株豬源性鏈球菌的分離鑒定及藥敏試驗[J];中國公共衛(wèi)生;2006年10期
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
1 吳曉e，
本文編號：2117808
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2117808.html
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