Qβ噬菌体A_2基因的克隆与生物活性分析

Cloning and Analyzing Biological Activity of A2 Gene in Qβ Phage

目的通过基因重组技术构建Qβ噬菌体A2基因表达载体,并对其生理学活性进行分析。方法采用PCR技术从Qβ基因组中扩增A2基因,克隆到pBAD表达载体中构建pBADA2重组质粒;对重组质粒进行酶切鉴定、DNA测序分析;转化宿主细胞JM109,SDS-PAGE检测Arabinose诱导后A2蛋白的表达;光电比浊法测定大肠杆菌生长曲线鉴定pBADA2在不同宿主细胞中的溶菌性。结果经菌落PCR筛选、序列测定和酶切鉴定证实表达载体pBADA2构建成功,并在JM109中获得高表达,其表达水平随Arabinose的浓度增加而增高,Arabinose在0.2%时达到高峰。OD660显示pBADA2具有溶菌性,可快速溶解大肠杆菌JM109、HB101和594,而对BE110没有溶解活性。结论构建成功高效表达A2蛋白的重组载体pBADA2,表达的蛋白具有良好的溶菌性,为新型抗菌药物开发奠定了理论和实践基础。

Objective To construct A2 gene expression vector in Qβ phage by gene recombination technology, and then analyze its physiological activities. Methods Amplified A2 gene in Qβ genome by PCR, cloned it into pBAD-24 expression vector to construct pBAD A2 recombinant plasmid. The recombinant plasmid was identified by restrictive enzymes digestion and DNA sequencing, then to be transfeeted into host cell JM109. After induced by Arabinose, the expression level of A2 was detected by SDS -PAGE. The growth curve of E.coli was obtained by phototurbidometry to test the baeteriolysis activity of pBADA2 in various host cells. Results After certified by PCR screening, DNA sequencing and restrictive enzymes digestion, the expression vector of pBADA2 was successfully constructed. The gene expression level is high in JM109 and related with Arabinose concentration, which reach its peak when Arabinose is 0.2%. OD660 value demonstrates that pBADA2 has the function of baeteriolysis, which could dissolve JM109,HB101 and 594 in E. eoli rapidly, but not BE110. Conclusion The highly expressed vector pBADA2 was successfully constructed. The protein expressed has the ideal function of baeteriolysis. All of these provide theoretical and practical bases for developing new anti - bacteria drugs.