鸡β防御素13基因的克隆、表达及其对猪源致病菌的抑制作用研究

Cloning and expression of chickenβ-defensin 13 gene and its inhibiting effect against pig-derived pathogens

为了探究鸡β防御素13(Gal-13)基因的克隆表达及对常见猪源致病菌的抑制活性,试验采用原核表达的方法,利用RT-PCR法,使用试剂盒从鸡肝脏组织中进行Gal-13基因的克隆,利用原核表达载体pGEX-4T-1构建重组表达质粒,再将重组质粒转化大肠杆菌TOP10诱导表达获得目的蛋白,并对诱导条件进行优化,然后采用牛津杯法对目的蛋白进行体外抑菌试验,检测目的蛋白对猪源大肠杆菌、金黄色葡萄球菌、副猪嗜血杆菌及链球菌的抑制活性。结果表明:克隆表达得到Gal-13基因,构建得到pGEX-4T-1-Gal-13表达质粒,转化大肠杆菌TOP10诱导表达,目的蛋白以包涵体形式存在,最佳诱导表达温度为37℃,最佳诱导表达时间为5 h,IPTG最佳诱导浓度为0.3 mmol/L,该蛋白对猪源大肠杆菌、金黄色葡萄球菌、副猪嗜血杆菌及链球菌均有一定抑制作用,平均抑菌圈直径分别为1.25,1.09,1.03,1.45 cm。说明Gal-13能够利用原核表达系统在大肠杆菌TOP10中诱导表达,且对4种猪源致病菌有一定的抑制作用。

In order to explore the cloning and expression of chickenβ-defensin 13(Gal-13)gene and its inhibiting effect against common pig-derived pathogens,the prokaryotic expression method was used in the experiment.First,RT-PCR method was used to clone the Gal-13 gene from chicken liver,and a recombinant expression plasmid was constructed using the prokaryotic expression vector pGEX-4 T-1;and the recombinant plasmid was transformed into Escherichia coli TOP10 to induce the expression to obtain the target protein,and induced conditions were optimized;and then an in vitro antibacterial test was carried out on the target protein by the Oxford cup method to explore the inhibitory activity of the target protein against pig-derived Escherichia coli,Staphylococcus aureus,Haemophilus parasuis and Streptococcus.The results showed that the Gal-13 gene was cloned and obtained,and the pGEX-4 T-1-Gal-13 expression plasmid was constructed,and was transformed into Escherichia coli TOP10 to induce the expression.The target protein existed in the form of inclusion bodies.The optimal inducible expression temperature was 37℃,the optimal induction time for expression was 5 h,and the optimal induction concentration of IPTG was 0.3 mmol/L.The protein had certain inhibiting effect against pig-derived Escherichia coli,Staphylococcus aureus,Haemophilus parasuis and Streptococcus.The average inhibition zone diameter was 1.25,1.09,1.03,1.45 cm,respectively.The results suggested that Gal-13 could be induced and expressed in Escherichia coli TOP10 via the prokaryotic expression system,and it had certain inhibiting effect against 4 kinds of pig-derived pathogens.