Cloning,Prokaryotic Expression and Protein Purification of PiNLP30 Gene of NLP Family in Phytophthora infestans
ZHU Li-dan
ZHU Jie-hua
ZHAO Dong-mei
YANG Zhi-hui
XU Jin
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.
Keywords：Phytophthora infestansPiNLP30 genesequence analysisprokaryotic expressionprotein purification
Publication Date：2014-01-01
Online Publishing Date：2026-09-12(First online date of this platform, not the publication date of the document)
Pages：5( 74-78 )