幽门螺杆菌黏附素BabA的大肠杆菌克隆构建及BabA黏附活性评估
Construction of the E.coli clone expressing adhesin BabA of Helicobacter pylori and evaluation of the adherence activity of BabA.
作者信息
Bai Yang, Chang Shao-hong, Wang Ji-de, Chen Ye, Zhang Zhao-san, Zhang Ya-li
机构信息
Institute of Digestive Diseases of PLA, First Military Medical University, Guangzhou 510515, China.
出版信息
Di Yi Jun Yi Da Xue Xue Bao. 2003 Apr;23(4):293-5, 309.
OBJECTIVE
To construct a recombinant E.coli strain that highly expresses blood group Ag-binding adhesin (BabA) of Helicobacter pylori (Hp) and to assess the adherence activity of Hp BabA.
METHODS
The gene fragment encoding BabA was amplified from Hp chromosomal DNA by PCR technique and inserted into prokaryotic expression vector pET-22b (+), which was then transformed into BL21 (DE3) E.coli strain for the expression of BabA recombinant protein. The adherence activity of Hp BabA obtained was assayed by counting under light microscope.
RESULTS
DNA sequence analysis showed that the sequence of babA2 DNA was in agreement with that published in GenBank. The BabA recombinant protein amounted to 34.8% of the total protein of the bacterium after IPTG induction for 3 h at 37 degrees Celsius, and BabA-mediated adherence was confirmed in vitro.
CONCLUSION
A clone expressing biologically active Hp BabA has been obtained, which may facilitate further study of the function of the adhesin.
目的
构建高效表达幽门螺杆菌（Hp）血型抗原结合黏附素（BabA）的重组大肠杆菌菌株，并评估Hp BabA的黏附活性。
方法
采用PCR技术从Hp染色体DNA中扩增编码BabA的基因片段，将其插入原核表达载体pET-22b(+)，然后转化至BL21(DE3)大肠杆菌菌株中表达BabA重组蛋白。通过光学显微镜计数来检测所获得的Hp BabA的黏附活性。
结果
DNA序列分析表明，babA2 DNA序列与GenBank中公布的序列一致。在37摄氏度用IPTG诱导3小时后，BabA重组蛋白占细菌总蛋白的34.8%，并且在体外证实了BabA介导的黏附作用。
结论
已获得表达具有生物活性的Hp BabA的克隆，这可能有助于进一步研究该黏附素的功能。
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