Gene Clone And Bioinformatics Analysis On Peptidoglycan-associated Lipoprotein A Of Haemophilus Parasuis

Haemophilus parasuis （HPS） is a member of Pasteurellaceae family and a genus of hemophilic Gram-negative bacteria. It is considered the main pathogen of the Claesser’s Disease. Researches indicate that HPS often causes the immunosuppression of organism when mixed infected with the pathogen of porcine reproductive and respiratory syndrome and as a result, the infection is aggravated. It brings great economic losses and has become a serious threat to the healthy development of pig industry. As one of the most important virulence factors of the HPS, the Peptidoglycan-associated lipoprotein（PalA） which is combined with peptidoglycan and cell membrane lipoprotein, widely exists in the Gram-negative bacteria and can be used as the vaccine candidate protein. In this research, the HPS type4SiChuan isolates（SC HSP4）, SC HPS5and SC HPS NT is applied. We use PCR technology for the amplification of HPS PalA gene and build its cloning vector for bioinformatics analysis.I. Isolation and identification of HPSUse the lung of the suspected HPS infected pig of the model farm in Suining, Sichuan as diseased tissue. After aseptic processing, coating it on the TSA medium and then choice the colony which has similar morphology with the HPS. After three times passaged purification, morphology, staining and culture characteristics testing, biochemical tests, serum-type testing and other basic biological characteristics tests are conducted.II. Cloning and sequence analysis of PalA geneWith the reference of the PalA gene nucleotide sequence CP001321.1, we use the Primer Premier5.0software to design and synthesize a pair of primers and use PCR technology for the amplification of HPS PalA gene and build its cloning vector. The HPS type4SiChuan isolates（SC HSP4）, SC HPS5and SC HPS NT which is isolated from the pathology is applied in our research. The results show that the nucleotide of the HPS PalA gene open reading frame is462bp in length, compared with the announced PalA gene nucleotide sequence CP001321.1, the resemblance is98%; the encoded amino acid is154aa in length, compared with the announced PalA gene encoded amino acid sequece YP₀02474727, the resemblance is97%.Ⅲ. Bioinformatics analysis of PalA gene encoded proteinThe bioinformatics analysis of PalA gene encoded protein of the HPS type5bacteria is conducted. The results show that its molecular weight equals16.4kDa, the theoretical isoelectric point PI value equals6.28and the instability index（II） equals33.62, indicating that the protein is stable, this protein has a obvious hydrophobic peak so it is hydrophilic. In PalA protein, there are possible transmembrane domains inside-out and outside-in respectively, the signal peptide splice site of the HPS PalA gene encoded protein is positioned between the18th and19th amino acid. We construct the3D structure of the HPS-PalA protein with the pal protein structure of haemophilus influenzae which has a resemblance up to68.61%as a template and through multiple sequence alignment and homology analysis, we find out that HPS-PalA exhibit high homology to the PalA gene of small actinomycetemcomitans, vice Haemophilus influenzae, Pasteurella haemolytica, they have a relatively close genetic relationship.