本文選題：型豬鏈球菌 + 原核表達��； 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,鑒定其免疫學特性。方法通過PCR從2型豬鏈球菌中國強毒株05ZYH33基因組中擴增基因Sao,構建重組表達質(zhì)粒pET28a-Sao,轉(zhuǎn)化E.coli BL21,異丙基硫代半乳糖苷誘導表達目的蛋白,十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳鑒定表達產(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殺菌作用。結(jié)果構建的重組質(zhì)�？梢栽谒拗骶懈咝П磉_,純化后獲得的重組蛋白純度可達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;全血殺菌實驗顯示,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,可明顯增強全血對細菌的殺傷作用。
[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)科大學檢驗醫(yī)學院生命科學學院;南京軍區(qū)軍事醫(yī)學研究所;
【基金】：國家自然科學基金(81172794;81071317;31170124) 江蘇省自然科學基金(BK2011098;BK2011097) 浙江省新苗人才計劃(2013R413039) 南京軍區(qū)醫(yī)學科技創(chuàng)新課題(ZX39)
【分類號】：R392

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