›› 2019, Vol. 46 ›› Issue (3): 661-668.doi: 10.16431/j.cnki.1671-7236.2019.03.003

• Biotechnology • Previous Articles     Next Articles

Cloning,Prokaryotic Expression and Bioinformatics Analysis of recN Gene of Pasteurella multocida

AZHANG Luyin, ZHENG Yiying, WANG Chengqiang, AN Qi, ZHANG Mengmeng, HUAGN Haifeng, ZHANG Zhenxing, LI Baobao, ZHU Shu, YANG Xiaojian, CAO Ruiyong, NIE Xin, DU Li, WANG Fengyang   

  1. Key Laboratory of Animal Genetic Engineering of Haikou City, Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Reseach of Hainan Province, College of Animal Science and Technology, Hainan University, Haikou 570228, China
  • Received:2018-07-03 Online:2019-03-20 Published:2019-03-20

PDF (PC)

156

Abstract:

This experiment was aimed to study the clone and prokaryotic expression of recN gene of Pasteurella multocida,and analyze its protein by bioinformatics method.A pair of primers was designed according to the recN gene sequence in GenBank(accession No.:CP003313.1),and the target gene fragment was obtained by PCR.The recombinant plasmid pET-28a(+)-recN was constructed and transformed into E.coli DH5α competent cells,and the plasmid was extracted for restriction enzyme digestion.The correct recombinant plasmid was transformed intoE.coli BL21(DE3) competent cells.The fusion protein was induced by IPTG,and identified by SDS-PAGE and Western blotting.The results showed that the recN gene with a size of about 1 677 bp was successfully cloned in this experiment.The size of the His-Tag fusion protein induced by expression was about 66.94 ku,and it mainly existed in the form of inclusion body.According to bioinformatics analysis,the recN protein had the molecular formula C2735H4428N786O855S16,the extinction coefficient was 24 785,the instability coefficient was 43.99,which was unstable protein;The theoretical isoelectric point (pI) was 5.62,which was acidic protein;The total average hydrophilicity was -0.316,which was the same hydrophobic protein as the experimentally expressed inclusion body protein;The half-life of reticulocytes in mammalian was estimated to be 30 h,the half-life in yeast and E.coli (in vivo) was more than 20 and 10 h,respectively.The secondary structure prediction was mainly alpha helix (64.87%) and random coil (21.00%);The hydrophobic analysis results showed that recN protein had 3 highly hydrophobic sexual regions and 9 highly hydrophilic regions.This results provided a reference basis for further exploration of the function of recNgene of Pasteurella multocida.

Key words: Pasteurella multocida; recN gene; clone; prokaryotic expression; bioinformatics analysis

CLC Number: