Construction of HlyA and HasA exocrine expression systems in Yersinia pestis

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.