多杀性巴氏杆菌中recN基因的克隆、原核表达及生物信息学分析

试验旨在对多杀性巴氏杆菌recN基因进行克隆和原核表达，并对其表达蛋白进行生物信息学分析。参照GenBank中recN基因序列（登录号：CP003313.1）设计1对引物，通过PCR扩增获得目的基因片段，构建pET-28a（+）-recN重组质粒并转化E.coliDH5α感受态细胞，提取质粒进行酶切鉴定，将鉴定正确的重组质粒转化E.coliBL21（DE3）感受态细胞，经IPTG诱导表达，对融合蛋白进行SDS-PAGE及Western blotting鉴定。结果表明，试验成功克隆了大小约为1 677 bp的recN基因序列，通过诱导表达的His-Tag融合蛋白大小约为66.94 ku，主要以包涵体形式存在。经生物信息学分析，recN蛋白的分子式为C₂₇₃₅H₄₄₂₈N₇₈₆O₈₅₅S₁₆，消光系数为24 785，不稳定系数为43.99，属于不稳定蛋白；理论等电点（pI）为5.62，为酸性蛋白；总平均亲水性为-0.316，与试验表达的包涵体蛋白性质相同，即同为疏水性蛋白；recN蛋白在哺乳动物网织红细胞的半衰期为30 h，在酵母（体内）中的半衰期>20 h，在大肠杆菌（体内）中的半衰期>10 h；二级结构主要以α-螺旋（64.87%）及无规则卷曲（21.00%）为主；经疏水性分析，预测该蛋白有3个高疏水性区域和9个高亲水性区域。本试验结果为进一步探究多杀性巴氏杆菌recN基因的功能提供了参考依据。

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

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.