| 无乳链球菌表面蛋白Rib基因克隆与重组表达 |
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| 引用本文： | 张荣,杜欣军,徐桂香,王菲,王硕.无乳链球菌表面蛋白Rib基因克隆与重组表达[J].动物医学进展,2012(3):24-28. |
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| 作者姓名： | 张荣 杜欣军 徐桂香 王菲 王硕 |
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| 作者单位： | 食品营养与安全省部共建教育部重点实验室天津科技大学食品工程与生物技术学院 |
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| 基金项目： | 天津市教委重点项目(2010ZD01) |
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| 摘 要： | 无乳链球菌是引起奶牛乳房炎的常见病原微生物之一,其表面蛋白作为一种高免疫原性的生物大分子,具有较高的种内特异性,是理想的候选疫苗与免疫检测靶标。本研究通过对无乳链球菌高免疫原性表面蛋白Rib的重组表达与抗体制备,为后期无乳链球菌的疫苗研制与检测奠定了良好的基础。首先提取了无乳链球菌基因组DNA,从中成功扩增出长度为452bp的编码表面蛋白Rib N端的基因片段。将这一片段连入重组表达载体pET-26b,转化受体菌株BL21(DE3)后利用IPTG进行诱导,结果表达了分子质量约为18ku的外源蛋白。在低温表达条件下,对IPTG诱导浓度进行优化,结果表明,IPTG的最适诱导浓度为0.05mmol/L。将诱导后的菌体进行超声波破碎并进行SDS-PAGE检测,结果显示重组蛋白以包涵体形式存在,对其进行变性、复性处理,利用亲和凝胶纯化后获得了纯度较高的重组蛋白。将重组蛋白免疫新西兰兔制备多克隆抗体,经检测抗体效价达到1∶480 000。
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| 关 键 词： | 无乳链球菌 表面蛋白 Rib基因 克隆与表达 |
Cloning and Recombinant Expression of Rib Gene of Streptococcus agalactiae Surface Protein |
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| ZHANG Rong,DU Xin-jun,XU Gui-xiang,WANG Fei,WANG Shuo.Cloning and Recombinant Expression of Rib Gene of Streptococcus agalactiae Surface Protein[J].Progress In Veterinary Medicine,2012(3):24-28. |
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| Authors: | ZHANG Rong DU Xin-jun XU Gui-xiang WANG Fei WANG Shuo |
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| Institution: | (Key Laboratory of Food Nutrition and Safety,Ministry of Education,Tianjin University of Science & Technology,Tianjin,300457,China) |
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| Abstract: | Streptococcus agalactiae caused mastitis in dairy cows is one of common pathogenic microorganisms.As a group of biomacromolecules with high immunogenicity,surface proteins have high intraspecific specificity,which are ideal vaccine components and targets for immunodetection.In this study,Rib surface protein which had high-immunogenicity was expressed.This study lays good foundation for S.agalactiae detection and vaccine development.A 452 bp fragment coding N-terminal of Rib surface protein was cloned from genomic DNA of S.agalactiae.This fragment was sub-cloned into pET-26b and expressed in E.coli BL21(DE3).After being induced by IPTG,a protein with molecular weight of 18 ku was expressed.At lower temperature,IPTG concentration was optimized,and the result indicated that 0.05 mmol/L IPTG was the optimal concentration for recombinant expression.Because the protein formed inclusion bodies,denaturation and renaturation procedures were introduced to get soluble protein.Purified protein was obtained using His-Bind Resin and polyclonal antibodies whose titers reached 480 000∶1 were prepared through immunizing rabbits. |
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| Keywords: | Streptococcus agalactiae surface protein Rib gene cloning and expression |
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