河南农业科学Issue(10)：74-78,5.
致病疫霉 NLP 家族基因 PiNLP30的克隆、原核表达及蛋白质纯化
Cloning,Prokaryotic Expression and Protein Purification of PiNLP30 Gene of NLP Family in Phytophthora infestans
摘要
Abstract
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.关键词
致病疫霉/PiNLP30 基因/序列分析/原核表达/蛋白质纯化Key words
Phytophthora infestans/PiNLP30 gene/sequence analysis/prokaryotic expression/protein purification分类
农业科技引用本文复制引用
朱丽丹,朱杰华,赵冬梅,杨志辉,徐进..致病疫霉 NLP 家族基因 PiNLP30的克隆、原核表达及蛋白质纯化[J].河南农业科学,2014,(10):74-78,5.基金项目
现代农业产业技术体系建设专项资金项目 （）
