Detection And Discrimination Of Virulent And Attenuated Strain Of Pseudorabies Virus Using Multiplex PCR And Microarray Chip And Primary Study On Capsular Polysaccharide Of Actinobacillus Pleuropneumoniae Serotype 2

The thesis is composed of parts describing three experiments1. Development of a multiplex PCR to differentiate PrV virulent and attenuated strain.Pseudorabies virus (PrV), a member of Alpha Herpesvirus, is the causative agent of Pseudorabies (Aujeszky's disease), one of the most serious infectious diseases of several domestic and wild animals. Swine is the natural host and reservoir of PrV. Pseudorabies is an economically important disease of swine industry in China. Currently it is an important, though difficult, task to prevent, control and eradicate this disease.To differentiate PrV wild type and vaccine virus, a multiplex polymerase chain reaction was developed and optimized to detect simultaneously pseudorabies virus (PrV) and to differentiate between field strain and gE mutant vaccine strain. Three pairs of specific primers were designed according to the conservative sequences of PrV gB ,gD and gE genes after alignments. The wild type and attenuated PrV could be detected by the multiplex PCR using three primer sets. Three expected specific bands of PrV gB gene (549 bp), gD gene (429 bp) and gE gene (366 bp) were all amplified from wild type virus while gE gene (366 bp) could not be amplified from PrV gE mutant. As little as 756 pg of PrV or 106 pg of TK~-/gI~-/gE~- PrV were obtained by the multiplex PCR. These results indicated that the multiplex PCR could be used to detect PrV field strain and gE mutant.2. Development of a microarray chip to discriminate PrV virulent and attenuated strain.This study discribes the development of a DNA microarray to improve the sensitivity in detection of PrV. The DNA segments that were printed on the microchip consisted of conservative gene fragments of PrV gD, gB, gE(gE~-, gE), pocine respiratory and reproductive symptome virus (PRRSV) ORF6, porcine circovirus type II (PCV2) ORF2, porcine parvovirus (PPV) NS1 and Japanese encephalititis virus (JEV) NS1, while human β-actin and porcine Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) gene fragments were used as internal controls.The presence of the specific genes encoding the conservative glycoprotein gD, gB, gE of wild type and gE-deleted strain of PrV were monitered by multiplex PCR followed by hybridization of denatured PCR products to the gene specific probes on the microchip. Both PrV strains showed positive hybridization signals of gD and gB gene, however, positive hybridization signal of the deleted domain of gE gene observedin wild type strain but not in gE mutant. Neither could cross reaction be demonstrated when fluorescence !abeled-PCR products of PrV hybridized with capture probes of PRRSV, JEV, PCV2 and PPV, nor did those of PPV and PCV when hybridized with PrV capture probes. The detection limit of the assay was 3.24 fg of DNA template, 10-fold higher sensitivity that normal gD-PCR. Our results suggest that microarray analysis may be very useful for detection of PrV and differentiation between field strain and gE mutant vaccine strain.3. Clone and expression of the capsular polysacchride derived from Actinobacillus pleuropneumoniae serotype 2.Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, which is a highly contagious respiratory disease. The disease is one of main contagious diseases affecting swine industry worldwide and causes tremendous economic loss to the swine industry. The capsular polysaccharide (CP) of A.pleuropneumoniae is required for virulence of the bacteria in swine, moreover, the type or quantity of CP affects A. pleuropneumoniae virulence.In this test, CpsA^ CpsB and CpsC genes of A.pleuropneumoniae serotype 2 (App2) were amplified by PCR. The sizes of amplicons were 1137bp, 429bp and 1146bp, respectively. These amplified products were then cloned into PMD18-T for sequence analysis and into expression plasmid pGEX-KG for protein expression. Recombinant CpsA, CpsB and CpsC protein were expressed in E.coli BL21 and were detected by SDS-PAGE and Western blotting. The reaction with swine poiyclonal serum against A. pleuropneumoniae was observed in CpsC, but not in CpsA and CpsB. In addition, for purpose of further exploring the functions of CpsA, CpsB and CpsC protein in mammalian cells, by using pcDNA3.1(+), three' recombinant expression plasmids, designated as pcDNA -CpsA^ pcDNA -CpsBn pcDNA -CpsC were constructed and enzymatically identified. These results paved the way for further investigation of CP.