Qβ噬菌体 A2 基因的克隆与生物活性分析

Cloning and analyzing biological activity of A2 gene in Qβ phage

关键词:  Qβ噬菌体  A2 基因  溶菌性

Key words:  Qβ phage  A2 gene  bacteriolysis activity

摘要:

［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pBAD A₂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pBAD A₂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pBAD A₂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pBAD A₂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pBAD A₂，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。

Abstract:

［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 transfected 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 bacteriolysis 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 bacteriolysis, which could dissolve JM109、HB101 and 594 in E.coli rapidly, but not BE110.［Conclusions］The highly expressed vector pBADA₂ was successfully constructed. The protein expressed has the ideal function of bacteriolysis. All of these provide theoretical and practical bases for developing new anti-bacteria drugs.