致病疫霉 NLP 家族基因 PiNLP30的克隆、原核表达及蛋白质纯化

摘要

为了制备致病疫霉 NLP 家族中 PiNLP30蛋白的多克隆抗体，根据 GenBank 中 PiNLP30的cDNA 序列及原核表达载体 pET28b 中的多克隆位点设计引物，对 PiNLP30基因进行 RT PCR 扩增和重组质粒 pET28b PiNLP30的构建，将重组质粒在大肠杆菌 BL21（DE3）表达体系中进行诱导表达，通过 SDS PAGE 检测蛋白质表达，并利用镍琼脂糖亲和层析进行蛋白质纯化。克隆和序列分析显示，该基因 cDNA 全长714 bp，编码237个氨基酸。该基因编码蛋白质是一个主要由不规则卷曲组成的亲水蛋白，含有一段19个氨基酸的信号肽序列。预测蛋白质分子量为26·6 kD，等电点5·49。经PCR、酶切及测序验证，成功构建了原核表达载体 pET28b PiNLP30，使用0·6 mmol/L IPTG 于37℃下诱导6 h 后蛋白质大量表达，表达的蛋白质主要以包涵体形式存在。经镍琼脂糖亲和层析进行纯化，获得了纯化的 PiNLP30蛋白，其质量浓度约为0·3 mg/mL。%The aim of this study was to express PiNLP30 of NLP family from Phytophthora infestans in prokaryotic cell,to provide a basis for preparation of PiNLP30 polyclonal antibody. The primers were designed according to the cDNA sequence of PiNLP30 from GenBank and the multiple cloning sites in prokaryotic expression vector pET28b,and the full length cDNA of PiNLP30 was amplified by RT-PCR,which was further cloned into vector pET28b.The recombinant plasmid pET28b-PiNLP30 was transformed into BL21 (DE3 ).The expression product of PiNLP30 was identified by SDS-PAGE and purified with Ni+ NTA affinity column. Sequence analysis indicated that the full-length cDNA was 714 bp,encoding a protein of 237 amino acids.The protein encoded by this gene was a hydrophilic protein mainly composed of irregular coil,with a signal peptide of 19 amino acids.The predicted molecular weight of the protein was 26·6 kD and had an isoelectric point of 5·49.The recombinant protein was expressed with 0·6 mmol/L IPTG induction for 6 h at 37 ℃,which mainly existed in inclusion body form.The purified recombinant PiNLP30 protein was obtained by Ni+ NTA affinity purification,with mass concentration of 0·3 mg/mL.