海参溶菌酶枯草芽孢杆菌基因工程菌构建

Construction of Genetic Recombination for Sea Cucumber Lysozyme in Bacillus subtilis

通过基因重组等技术构建重组海参溶菌酶的枯草芽孢杆菌基因工程菌,并对此基因工程菌进行生长曲线的测定和稳定性分析。结果表明,海参溶菌酶基因特异引物在400 bp处扩增出特异性条带,与预期的海参溶菌酶基因大小一致;重组表达质粒pHT43-SjLys经双酶切验证得8 000 bp和400 bp左右的片段;重组海参溶菌酶的枯草芽孢杆菌基因工程菌与原始菌株WB600相比生长趋势基本一致,外源基因的插入对菌体的生理代谢未造成太大影响;在无选择压力的条件下,重组质粒的稳定性良好,连续传5次后的遗传稳定性为94%,并且提取质粒和双酶切验证后发现工程菌没有发现重排或丢失现象。表明重组海参溶菌酶基因工程菌pHT43-SjLys/WB600构建成功。

The expression of lysozyme from sea cucumber Stichopus japonicus in Bacillus subtilis were constructed by the method of recombinant DNA technique, and the expression plasmid pHT43-SjLys of growth curve and stability were analyzed. Results showed that a specific strip about 400 bp was visible in the result of agarose gel electrophoresis, which was consistent with expected size of sea cucumber lysozyme gene. Construction of the lysozyme was performed in a vector pHT43 and validated by double digestion with BamHⅠand SmaⅠ, witch indicated the fragment size was consistent with the expected result. The growth trend of engineering bacteria was consistent compared with the wild type strain WB600, and insertion of foreign genes did not affect the cell’s metabolism. In the absence of selection pressure, the recombinant plasmid was stability, and there was no gene rearrangement and lost. The results showed that the recombinant sea cucumber lysozyme genetic engineering bacteria pHT43-SjLys/WB600 is constructed successfully.