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H. parasuis disease caused by Haemophilus parasuis(H. parasuis) are widely spread in many countries, and has become one of the most important bacterial diseases threatening pig industry in the world. In recent years, H. parasuis disease with increasing incidence have occurred successively in various provinces and cities of china, and caused some problems to pig industry. Serotypes of H. parasuis arecomplex and diverse, and have a lot of unidentified serotypes except 15 identified serotypes. There were greatly different pathogenicity between different serotypes of H. parasuis. The general view was that 1, 2, 4, 5, 8, 10, 12, 13, 14 and 15 serotypes are virulent, and 3, 6, 7, 9 and 11 serotypes are non-virulence types. Although the genome of H. parasuis has been publicated (GenBank: Acc. CP001321; GenBank Acc. ABKM00000000), but virulence factors of H. parasuis are still poorly known. The researches on mechanism of pathogenesis and immune of Gram-negative bacteria of outer membrane proteins have made great progresses, as well as the research on outer membrane proteins and virulence of H. parasuis have got attention, too. The outer membrane protein P2 (ompP2) has been proved to be a pathogenic virulence factors of Haemophilus influenza which is the same genus with H. parasuis. However, whether the ompP2 is also a virulence factor of H. parasuis need further study.The objective of this study was to further analysis the gene structure features of H. parasuis ompP2, to compare the effect of ompP2 with different structural features on cell, thus to explore the biological functions of genes. The results were as follows:1 The structural features of H. parasuis ompP2 gene1.1 There were two gene structural types of H. parasuis ompP2OmpP2 gene of H. parasuis from 15 serotypes of reference and 19 strains isolated from organs of pigs suffering from H. parasuis disease were cloned and analyzed. The results showed the length of ompP2 gene in 15 kinds of serotyped reference strains were 1077 ~ 1203 bp encoding 358 ~ 400 length of amino acids and proteins of 38.49 ~ 43.35kDa, compared with non-toxic serotypes (3, 6, 7, 9 and 11) reference strains, ompP2 genes from reference virulence serotypes (2, 4, 5, 10, 12, 13,14 and 15) strains were all showed two pieces of consecutive base deletion, except serotypes 1 and 8 reference strains. These base deletions presented respectively in the regions of 450 ~ 524bp and 770 ~ 844bp, a total of 100bp all so. Meanwhile, there were still more point mutations among this gene of different serotypes. The ompP2 genes from 19 clinical isolates were 1080 ~ 1086 bp, predictedly encoding 358 ~ 361 amino acids, proteins of 38.38 ~ 38.91kDa. It was mentioned that those clinical isolates presented the same continuous two base deletions as virulent strains.1.2 The structures of proteins encoded by H. parasuis ompP2 gene were different in different strainsThe feature of transmembrane structure and surface exposed loops were analyzed by using PRED-TMBB software(http://bioinformatics. biol. uoa. gr/PRED-TMBB/and http://bio.dfci.harvard. edu/Tools/antigenic.pl )and it was found that 19 clinical isolates and reference strains of serotype 2, 4, 5, 10, 12, 13, 14 and 15 had eight exposed surface loops, but the serotypes 1, 3, 6, 7, 8, 9 and 11 had nine exposed surface loops, the former lacked Loop9 between Loop4 and Loop5. The exposed epitopes of outer membrane proteins encoded by ompP2 gene of 19 clinical isolates and 15 reference strains were analyzed, and it was found that there were 5 expoesd epitopes in 19 clinical isolates, and reference strains which serotypes were 1, 5, 8, 13, 14 and 15, 2 exposed epitopes in serotype 7, 6 exposed epitopes in serotype 3, 4, 10 and 12, 4 exposed epitopes in serotype 6, 7 and 9, 3 exposed epitopes in serotype 11. Interestingly, two consecutive base deletions of ompP2 gene increased the number of exposed epitopes. OmpP2 gene of serotype 1 and 8 encoded 5 exposed epitopes although they hadn’t the two pieces of base deletions. It is always belived that the numbers(5～7) of extracellular epitopes encoded by ompP2 genes of virulence serotyps 1, 2, 4, 5, 8, 10, 12, 13, 14 and 15 were more than those of non-virulent serotypes 6, 7, 9 and 11 (3 ~ 4). There was a membrane surface epitope VTDQ (K) ALGVGL, which located in Loop8, in 19 clinical isolates and all the virulent reference strains, not in non-virulent reference strains. There was a surface epitope EKIDFVRT, which located in Loop4, in all clinical isolates and virulent reference strains except serotype 1 and 8.2 The correlation between structure and virulence of H.parasuis ompP2 geneEukaryotic expression vectors of two types of ompP2 gene structure for ompP2 gene of H. parasuis were constructed in this research. They were used to transfected Marc145 cell lines to study the correlation between structure and virulence of ompP2 gene. Cell morphological changes of transfected Marc-145 cell lines were observed. The results showed that eukaryotic expression vectors containing two different ompP2 genes caused obviously different cytotoxicity to Marc-145 cell lines. OmpP2 gene from Serotype 5 took obvious cytotoxicity to Marc-145 cell lines, and the cytotoxicity was enhanced with the increase of plasmid dose, showing a clear dose-effect relationship. Eukaryotic expression vector containg ompP2 gene from serotype 11 had no significant cytotoxicity to Marc-145 cell lines under the same transfection dose and conditions. It was first reported there was close relationship between the structure of ompP2 gene and virulence of H.parasuis, ompP2 gene missing two pieces of consecutive bases was a virulent gene of H.parasuis.3 DNA vaccine of H. parasuis ompP2 gene had protective effectMale Balb / c mice were immunized with DNA vaccine made of H.parasuis ompP2 (PcDNA3-ompP2-5) in this study, and were infected with serotype 5 H.parasuis. The results showed that H.parasuis DNA vaccine could delay the death time of mice when the immune dose was 150μg / one or 100μg / one adding 603 adjuvant, DNA vaccine could delay the death time of mice after infected H.parasuis, which indicated that the vaccine has a protective effect .4 The establishment of double PCR method for H. parasuis ompP2 and 16SrRNA detectionA pair of specific primers were designed and amplied to detect the base deletion region of ompP2 gene from H. parasuis in this research. Fifteen kinds of reference serotypes and 81 clinical isolates were detected by the above primers. The two consecutive base deletion regions could be amplified in all clinical isolates. The concentrations of ompP2 gene and 16SrRNA gene primers and double PCR reaction conditions were optimized to amplify deletion region gene of ompP2 gene and 16SrRNA gene. Fifteen kinds of reference serotypes and 81 clinical isolates were detected by the above double PCR. Because 16SrRNA primers can specifically identify H. parasuis, and the base-deletion-region specific primers based on ompP2 gene deletion region can distinguish ompP2 gene missing or not, combining these two pairs of primers will expect to create a double PCR detection method targeting at ompP2 and 16SrRNA genes to distinguish virulence and non-virulence H. parasuis isolates except serotype 1and 8.
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