副猪嗜血杆菌分离株hhdA和hhdB基因的克隆及同源性分析

用预先设计好的hhdA和hhdB基因的PCR扩增引物，扩增不同血清型的副猪嗜血杆菌分离株中的hhdA和hhdB基因，将扩增产物与克隆载体pMD ^TM18-T连接，转化入感受态细胞DH5α，通过PCR、双酶切及序列测定，测序结果表明：扩增的16株HPS菌中有11株能扩增出hhdA基因，其中10株有测序结果，该10株菌的hhdA基因全基因序列有7种长度，分别为1754、1755、1758、1759、1760、1761和1762bp。另外，16株HPS菌中有9株能扩增出hhdB基因，该9株菌的hhdB基因全基因序列有7种长度，分别为1628、1637、1639、1640、1641、1646和1647bp。对于相同血清型的不同菌株来说，它们的2段目的基因序列长度两两不一致。强毒株中，71％的菌株均能扩增出hhdA和hhdB基因；中毒型菌株也基本可以扩增出hhdA和hhdB基因；而无毒型菌株均没有扩增出目的基因。同源性比对可知：所分离菌株的hhdA之间和hhdB基因之间，以及它们分别与GenBank中副猪嗜血杆菌SH0165的hhdA和hhdB基因之间序列同源性都在98％以上，说明所分离的菌株中均含有hhdA和hhdB基因序列。同源系统进化树分析可知，hhdA和hhdB基因在分离菌菌株间具有良好的保守性，可作为研制HPS新型疫苗的候选基因。

The cloning and homology analysis of gene hhdA and hhdB from isolated strains of Haemophilus Parasuis

Two pairs of previous designed PCR primers were used to amplify gene hhdA and hhdB from isolated strains of different serotype Haemophilus parasui. The PCR products were jointed with the cloning vector pMDTV18-T,and transformed with competent cell DHSa. After the trans- formed products were double digested and complete sequence were determined,the results indica- ted that 11 of 16 strains HPS exist gene hhdA, 10 of this 11 strains were sequencing and 7 se- quence lengths were detected in this 10 strains. They were 1 754,1 755,1 758,1 759,1 760,1 761 and 1 762 bp,respectively. Besides,9 of 16 strains HPS exist gene hhdB and 7 sequence lengths were detected. They were 1 628,1 637,1 639,1 640,1 641,1 646 and 1 647 bp,respectively. Among the same serotype HPS strains,the length of the two gene were different. In virulent strains,71% were amplified gene hhdA and hhdB; In mesogen strains,the two genes were mainly exist,but no existence in avirulent strains. Homology comparison indicated that gene hhdA and gene hhdB between i- solated strains and HPS SH0165 strain in GenBank were respectively above 98% homology, indicating that gene hhdA and hhdB indeed exist in isolated strains,and have good conservatism.