嗜虫耶尔森氏菌HlyA及HasA外分泌表达系统的构建

Construction of HlyA and HasA exocrine expression systems in Yersinia pestis

摘要:

[目的]构建一株具备外分泌蛋白功能的工程菌，解决杀虫毒素无法由胞内分泌至胞外，无法直接作用于虫体等问题，为松墨天牛防治提供新思路。[方法]本研究先测定从松墨天牛肠道及其生境中分离出的嗜虫耶尔森氏菌（CSLH88）的生长特性及抗性，进而对其进行分子改造。构建HlyA（pGHKW2）以及HasA（pGHKW4）外分泌表达载体，利用电穿孔法将其转入CSLH88菌株，获得能够表达绿色荧光蛋白的工程菌。利用稀释涂板及荧光体式镜检测技术对两个质粒进行遗传稳定性检测，并采用SDS-PAGE及Western blotting技术验证蛋白外分泌功能。[结果]CSLH88菌株培养2-4 h能够进入对数生长期，并对卡那霉素（Kan）敏感。成功构建了含有Kan抗性基因的pGHKW2（GenBank：MK562405）和pGHKW4（GenBank：MK562404）两个外分泌表达载体的CSLH88工程菌株。其中，发现pGHKW4质粒更加适合在嗜虫耶尔森氏菌中稳定遗传。SDS-PAGE及Western blotting检测结果表明HlyA系统无法在CSLH88菌株中将目的蛋白分泌到胞外，而HasA系统则可以有效地发挥外分泌表达功能。[结论]通过对HlyA及HasA两个外分泌表达系统进行研究，从中筛选出HasA型血红素转运系统作为CSLH88菌株的外分泌表达系统，为后续外分泌杀虫毒素蛋白菌株构建以及CSLH88菌株的致病性研究奠定基础。

关键词:松墨天牛;嗜虫耶尔森氏菌;外分泌表达系统;绿色荧光蛋白

Abstract:

[Objective] Construction of transgenic strain can effectively improve the insecticidal effect. But the insecticidal toxin cannot be secreted from intracellular to extracellular, so it cannot directly act on the insect body. Therefore, if we can construct an engineered strain with exocrine protein function, it is an effective way to solve these problems.[Methods] In this study, the growth characteristics and resistance of Yersinia pestis (CSLH88) isolated from the intestinal tract and habitat of M. alternatus were determined, and then the molecular modification was carried out. The exocrine expression vectors of HlyA (pGHKW2) and HasA (pGHKW4) were constructed and transformed into CSLH88 strain by electroporation to obtain engineering strain capable of expressing green fluorescent protein. The genetic stability of the two plasmids was tested, and the exocrine function of the proteins was verified by SDS-PAGE and Western blotting techniques. [Results] The strain of CSLH88 could enter logarithmic growth stage after 2-4 hours of culture and had no resistance to kanamycin (Kan). Two exocrine expression vectors, pGHKW2 (GenBank:MK562405) and pGHKW4 (GenBank:MK562404), were successfully constructed. It was found that pGHKW4 plasmid was more suitable for stable inheritance in Y. pestis. The results of SDS-PAGE and Western blot showed that the secretion of extracellular proteins in HlyA system protein was blocked, while the HasA system could play an exocrine expression function in CSLH88 strain. [Conclusion] By studying the exocrine expression systems of HlyA and HasA, HasA heme transport system was selected as the exocrine expression system of CSLH88 strain. It lays a foundation for the construction of exocrine toxin protein strain and the pathogenicity of CSLH88 strain.

Key words:Monochamus alternatus Hope;Yersinia pestis;exocrine expression system;green fluorescent protein