番茄叶绿体小分子热激蛋白多克隆抗体的制备

The Production of Polyclonal Antibody of the Chloroplast Small Heat Shock Protein in Tomato

目的为深入研究叶绿体sHSP的功能,构建有效的原核表达体系,制备高特异性的叶绿体sHSP的多克隆抗体。方法将PCR方法得到的番茄叶绿体小分子热激蛋白基因(cpshsp)全长cDNA基因片段,克隆入融合表达载体pGEX6p-1中,在强启动子作用下在大肠杆菌E.coli BL21(DE3)中诱导表达,并将sHSP纯化产物免疫新西兰大白兔,western blot检测sHSP多克隆抗体。结果表达菌株经0.2 mmol/L IPTG诱导4h后,GST-CPsHSP形成包涵体,sHSP纯化产物免疫新西兰大白兔后得到叶绿体sHSP多克隆抗体,western blot检测表明该抗体的特异性和效价较好。结论构建的原核表达载体pGEX6p-1/cpshsp,在一定的诱导条件下,在DE3中得到稳定高效的融合表达,为制备叶绿体sHSP抗体提供足够的抗原,为进一步研究叶绿体sHSP的功能奠定了基础。

Objective：To further study the function of the chloroplast small heat shock protein（sHSP）, we inserted the gene of cpshsp of tomato into prokaryotic expression vector pGEX6p - 1 to obtain the high - specific sHSP - antibody. Methods ： Recombinant protein was expressed under the control of strong promoter in strain BL21 （ DE3 ） of E. coli. The purified protein was injected into a rabbit, the specificity and effect of polyclonal antibody against sHSP were analyzed by Western Blot. Results：The chloroplast sHSP mainly existed in the form of inclusion body when the strain was induced by 0. 2mmol/LIPTG for 4 hours. The anti - sHSP polyclonal antibody was raised in the rabbit and had higher specificity and effect. Conclusion：Given appropriated conditions, the recombinant prokaryotic expression vector pGEX6p - 1/ cpshsp can express the higher specific and effective polyclonal antibody, which layed a foundation to study the function of chloroplast sHSP.