幽门螺杆菌4种黏附素基因保守区的克隆、序列及其生物信息学分析

目的克隆幽门螺杆菌(Helicobacterpylori, Hp)4种黏附素(BabA、AlpA、AlpB和HopZ)的保守区,并对其进行序列及生物信息学分析,为研究Hp黏附素的分子机制和免疫原性提供实验基础。方法利用ANTHEPROT V4.3c 软件分析已证实的Hp4种黏附素蛋白序列,发现共同保守区(命名为CB),推导出其DNA序列,设计CB特异引物,将PCR产物定向插入pET-22b(+)载体,构建保守区的重组克隆。DNA自动分析仪进行序列测定,生物信息学软件对其生物学特性进行分析。结果构建了4种黏附素共同保守区的重组质粒,测序显示CB基因长588 bp,该基因编码195个氨基酸,与4种黏附素保守区的同源性均达50%以上,ANTHEPROT V4.3c软件预测其蛋白质相对分子质量约为22 500,并显示出了良好的抗原性和疏水性。BLAST分析了836 767个序列,与其同源性达40%的均为Hp序列。结论Hp4种黏附素存在同源性接近的保守区,表明其黏附作用可能有相似的分子基础,生物信息学分析表明其具有优良的免疫原性和严格的种属特异性。

Conservative region of the genes encoding four adhesins of Helicobacterpylori: cloning, sequence analysis and biological information analysis

Objective To clone the conserved regions of the genes encoding the 4 adhesins (BabA, AlpA, AlpB and HopZ) of Helicobacter pylori(H.pylori) and analyze their sequences and biological information, thus facilitating further research in the molecular mechanism and immunogenicity of H.pylori adhesins. Methods Common conserved region (designated as CB) was identified from the confirmed sequences (by ANTHEPROT V4.3c software package) of the 4 adhesin proteins. Their DNA sequences were deduced, according to which primers specific to CB were designed for subsequent PCR, and the products were inserted directionally into pET-22b(+) vector to construct recombinant clones of the conserved region. The DNA sequences were determined with the basic local alignment sequence tool (BLAST) and the biological properties analyzed with ANTHEPROT V4.3c software package. Results The recombinant plasmid containing the CB sequence was constructed. DNA sequencing showed an open reading frame of 588 bp in length, encoding 195 amino acids. The homogenicity of conservative region of the 4 adhensin genes was above 50%. The corresponding protein possessed a relative molecular mass (M r ) of 22 500 as predicted by ANTHEPROT V4.3c software prediction, with excellent antigencity and hydrophobicity. There were 836 767 sequences ana-lyzed with BLAST, in which those with homogenicity of 40% with the identified CB sequence were categorized into H.pylori se-quences. Conclusion There are conservative regions in the 4 adhesin genes with similar homogenicity, suggesting similar mole- cular basis for adhension of the adhesins. Biological information analysis indicates that CB has excellent immunogenicity and strict species specificity.