鸡β防御素2成熟肽基因在毕赤酵母中的表达及其抗菌活性分析

为构建基因重组酵母菌株表达鸡β防御素2(Gal-2)并研究其抗菌活性,将鸡β防御素2成熟肽基因克隆到pPIC9K中,获得重组分泌表达质粒pPIC9K-Gal-2并用电转化法转入酵母菌GS115,筛选出阳性克隆后用甲醇诱导,表达上清经Tricine-SDS-PAGE和抑菌试验检测,最后通过禽致病性大肠杆菌CVCC1565感染雏鸡,研究表达上清的预防效果。结果表明,重组分泌表达质粒pPIC9K-Gal-2构建成功,Tricine-SDS-PAGE检测到分子质量在5.8ku左右的目的条带,表达上清能抑制包括2种禽致病菌在内的多种细菌的生长;在禽致病性大肠杆菌感染雏鸡的试验中,注射不同浓度的Gal-2表达上清组与空酵母表达上清组、PBS对照组相比,死亡率均极显著降低(P<0.01),表达上清显示了较好的预防效果。该研究成功构建了表达鸡β防御素2的毕赤酵母菌株,为鸡防御素作为新型饲料添加剂的研发提供了基础。

Expression of Gal-2 mature peptide gene in Pichia Pastoris and analysis of antimicrobial activity of the recombinant protein

To construct the recombinant Pichia pastoris expressing chicken beta-defensin-2(Gal-2) and research the antimicrobial activity,the gene encoding Gal-2 mature peptide was cloned into an expression vector pPIC9K which was then transformed into P.pastoris GS115 by electroporation.The positive clones of recombinant yeast were screened and induced by methanol.The expressed supernatant was detected by Tricine-SDS-PAGE and antibacterial experiment.Finally,the prevention effect of expressed supernatant was researched by the experiment of chicken infected by Escherichia coli CVCC1565.The result showed that the recombinant secretion expression plasmid pPIC9K-Gal-2 was successfully constructed and the 5.8 ku chicken Gal-2 protein was detected by Tricine-SDS-PAGE.The expressed supernatant showed an inhibiting effect on the growth of many bacteria including two avian pathogenic bacteria of Salmonella Pullorum S2 and birds pathogenic E.coli CVCC1565.In the experiment of chicken infected by E.coli CVCC1565,compared to injection of empty yeast expression supernatant group and PBS control group,the mortality of Gal-2 of different concentration expression supernatant group was significantly decreased(P<0.01).The prevention effect of Gal-2 expressed supernatant reached high level.The recombinant P.pastoris GS115 was successfully constructed,which provided the basis for research and development of chicken defensin as new feed additives.