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| 牛源无乳链球菌CAMP因子(cfb)的克隆及原核表达 | |
| 引用本文： | 王向柳,王长法,杨宏军,阿合买提·买买提,高运东,仲跻峰.牛源无乳链球菌CAMP因子(cfb)的克隆及原核表达[J].新疆农业科学,2009,46(1). |
| 作者姓名： | 王向柳 王长法 杨宏军 阿合买提·买买提 高运东 仲跻峰 |
| 作者单位： | 1. 山东省农业科学院奶牛研究中心,济南,250100;新疆农业大学动物医学学院,乌鲁木齐,830052 2. 山东省农业科学院奶牛研究中心,济南,250100 3. 新疆农业大学动物医学学院,乌鲁木齐,830052 |
| 基金项目： | 山东省中青年科学家基金，山东省农科院重大成果培育项目，山东省农科院高技术自主创新基金 |
| 摘 要： | 提取能产生CAMP反应的牛源无乳链球菌山东分离株的基因组DNA,PCR扩增CAMP因子基因,重组到pMD18-T载体中。测序结果表明,扩增片断含有768 bp的ORF,可编码含255个氨基酸的成熟蛋白,与已报道的无乳链球菌CAMP因子蛋白的氨基酸组成相比,第8位Tyr→Cys,第95位Pro→Ser,第126位Thr→Ala的3个氨基酸发生了点突变,同源性为98.4%。成功构建原核表达载体pET32a+/cfb,SDS-PAGE分析蛋白质表达水平,IPTG诱导后表达的融合蛋白分子量为48 kD,表达量占菌体总蛋白的52.7%。这为进一步研究其致病与免疫机理、疫苗设计提供了条件。 |
| 关 键 词： | 无乳链球菌 CAMP因子 克隆 表达 |
Cloning and expression CAMP gene of Streptococcus agalactiae from mastitis milk |
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| WANG Xiang-liu,WANG Chang-fa,YANG Hong-jun,Ahemaiti·Maimaiti,GAO Yun-dong,ZHONG Ji-feng.Cloning and expression CAMP gene of Streptococcus agalactiae from mastitis milk[J].Xinjiang Agricultural Sciences,2009,46(1). | |
| Authors: | WANG Xiang-liu WANG Chang-fa YANG Hong-jun Ahemaiti·Maimaiti GAO Yun-dong ZHONG Ji-feng |
| Abstract: | CAMP factor gene was extracted from genome DNA of Streptococcus agalactiae by PCR,and the PCR product was inserted into vector pMD18-T.Sequence analysis showed that the Streptococcus agalactiae CAMP gene(cfb) was composed of 768 nucleotides encoding a mature polypeptide 255 amino acids.Compared with other Streptococcus agalactiae CAMP gene(cfb) reported shared the identity of 98.4%.The positive colony which were identified by digestion and PCR showed that the recombinant plasmid pET32a+/cfb was constructed successfully.SDS-PAGE result showed that recombinant protein,with a molecular weight of 48 ku,was expressed in inclusion bodies in E.coli and the recombinant protein accounted for 52.7% of the total proteins.This study provides conditions for the research of pathogenic and immune mechanism and vaccine design. |
| Keywords: | Streptococcus agalactiae CAMP factor cloning expression |
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