副猪嗜血杆菌血清4型不同菌株间毒力相关基因验证及LpxC基因生物信息学分析

Virulence-related Gene Verification and LpxC Gene Bioinformatics Analysis of Different Strains of Haemophilus parasuis Serotype 4

为探究副猪嗜血杆菌同一血清型不同菌株间毒力相关基因的差异以及LpxC基因的遗传特性,本试验设计35种毒力相关基因的特异性引物,对副猪嗜血杆菌血清型4型的两种菌株进行PCR验证,并将两株菌株的LpxC基因连接到T5载体上,转化到大肠杆菌感受态细胞中进行克隆及测序,得到921bp目的片段。在NCBI上进行比对分析,运用DNA star软件对LpxC基因进行核苷酸同源性比对,同时对LpxC基因编码的蛋白质进行生物信息学分析。结果表明:35种毒力相关基因中除SapA基因,其余34种在两个菌株中共同存在,两株HPS的LpxC基因序列与各参考菌株之间的同源性均在97%~100%之间,说明LpxC基因在副猪嗜血杆菌中保守存在。系统进化树结果显示XM1602菌株LpxC基因与SH0165菌株处于同一分支,FS0035菌株LpxC基因与ZJ0906菌株处于同一分支。同时,菌株FS0035和XM1602的LpxC基因编码的蛋白质等电点分别为5.59和5.45;该蛋白质为亲水性蛋白,无跨膜结构;二级结构包括α螺旋、伸展链、β转角和无规卷曲。结果证实,同一血清4型,两株不同的副猪嗜血杆菌菌株在毒力相关基因上存在一定的差异;LpxC基因在两个菌株中保守性较高,变异性较低。

In order to explore the differences in virulence-related genes between the different strains of the same serotype of Haemophilus parasuis and the genetic characteristics of the LpxC gene, we designed 35 specific primers for virulence-related genes.Two strains of Haemophilus parasuis type 4 were verified by PCR. The LpxC gene of the two strains was ligated into the T5 vector, transformed into E. coli competent cells for cloning and sequencing to obtain a 921 bp target fragment. The alignment analysis was performed on NCBI, and the nucleotide homology alignment of the LpxC gene was performed by DNA star software.The bioinformatics analysis of the protein encoded by the LpxC gene was performed. The results showed that among the 35 virulence-related genes, except for the SapA gene, the remaining 34 genes coexist in two strains. The homology of the LpxC gene sequences between the two HPS strains and the reference strains was between 97% and 100%.The LpxC gene was found to be conserved in Haemophilus parasuis. Phylogenetic tree results showed that the LpxC gene of XM1602 strain was in the same branch as SH0165 strain, and the LpxC gene of FS0035 strain was in the same branch as ZJ0906 strain. The isoelectric points of the proteins encoded by LpxC gene of FS0035 and XM1602 are 5.59 and 5.45. The protein is hydrophilic and has no transmembrane structure. The secondary structure includes alpha helix, stretching chain, beta corner and random curl. In conclusion, there were differences in virulence related genes between the two same sera 4 strains of Haemophilus parasuis. And the LpxC gene was highly conservative and had low variability in the two strains.