远缘链球菌中葡萄糖基转移酶催化活性区基因的克隆和初步表达

Cloning and expression of gene encoding catalytic region of glucosyltransferase from Streptococcus sobrinus 6715

目的 :构建一株高效表达Streptococcussobrinus 6715中GTF -I催化活性区 (含有B细胞表位 )多肽的菌株 ,为抗GTF -I的单克隆抗体的检测和防治龋病亚单位疫苗的研究奠定基础。方法 :提取基因组DNA ,然后用PCR技术扩增出中目的肽段的约 1.1kb编码基因 ,并将其克隆至表达载体 pGEX -4T -1中构建 pGEX -gtf表达质粒。该质粒转化大肠杆菌JM 10 9后获得重组表达菌株。PCR筛选阳性克隆子。异丙基-β -D -硫代半乳糖苷 (IPTG)诱导 ,确定含有 pGEX -gtf的大肠杆菌JM 10 9的表达情况。 结果 :含有质粒pGEX -gtf的大肠杆菌JM 10 9能够表达GST -GTF融合蛋白。 结论 :成功扩增目的基因 ,并且把它定向克隆到表达载体pGEX -4T -1中构建表达质粒 。

Objective: To establish a recombinant strain over-expressing catalytic region of glucosyltransferase(GTF-I,containing B-Cell epitope) from Streptococcus sobrinus 6715 for the study of anti GTF-I monoclonal antibody and the sub-unit of anti-caries vaccine. Methods: Expressing plasmid pGEX-gtf was constructed from the vector pGEX-4T-1 and the target PCR amplified product(1.1 kb) and then transformed into host strain Escherichia coli JM109. The recombinant strain harboring the pGEX-gtf plasmid was verified by PCR. The expression of the gene in the recombinant strain was also tested by SDS-PAGE before and after induction by isopropy-β-D-thiogalactosid(IPTG). Results: The strain expressed GST-GTF fusion protein.Conclusion: GTF-1 expressing plasmid can produce targeted protein in the prokaryote expressing system