Molecular Epidemiology Investigation Of PRRS In The Western Of Henan Province And The Construction And Identification For Recombinant ORF5Attenuated Salmonella

Porcine reproductive and respiratory syndrome (PRRS) was an importantinfectious disease in swine, caused by porcine reproductive and respiratory syndromevirus(PRRSV), which characterized with severe reproductive failure in sows, andrespiratory disease in piglets or growing pigs. Recently, a variat PRRSV carried moredifficulties in prevention and control for this disease. In recent year, the DNA vaccinecarried by attenuated Salmonella through the mucosa into organism has became moreand more popular. A host-vector balanced lethal system of Salmonella choleraesuisC78-1Strain was constructed by deleting asd gene based on the crpC78-1. Then thePRRSV dORF5gene was carried by this system to discuss the effection of the Host-Vector Balanced Lethal System and the new generation vaccine of PRRS. The mainresearch was described as follows:1. Molecular epidemiology investigation of PRRS in the western of Henan provinceTwenty one complete PRRSV ORF5s were amplified successfully from thesamples, and the determination for sequences indicated that the similarity of amongthese sequences was between96.5%and100.0%. Compared to JXA1, MLV, VR-2332and LV， the similarity of these sequences was96.6%~98.9%,88.4%~89.2%,88.0%~89.5%,66.8%~67.3%respectively. The part of Nsp2gene for mascclinesamples were also amplified, gene homology analysis was performed with Nsp2ofVR-2332deposited in GenBank after determination for sequences, and the two pointsand90bp bases deleted were found in all of the twenty one Nsp2genes. This studyidentified the vatiations of prevalent PRRSV for ORF5gene and Nsp2gene in thewestern of Henan province, and provided some basis for controlling PRRS in this area. 2. Construction and identification of host-vector balanced lethal system for mutant ofSalmonella choleraesuis C78-1The E.coli χ7213contained pREΔasd was conjugated with ΔcrpC78-1mutant,the ΔcrpΔasdC78-1mutant was successfully determined by PCR and sequencinganalysis after the two-step method introduced by the transduction of recombinantsuicide plasmid. The ability of growth for ΔcrpΔasdC78-1mutant in circumstancewithout DAP was lost; the ability of fermention for maltose, xylose, arabinose weremissed, but the mutant can also ferment glucose;315bp-deleted asd fragement can betransmited stablely; Compared to Salmonella choleraesuis C78-1, the serotype ofΔcrpΔasdC78-1mutant had no difference. Then the host-vector balanced lethal systemof Salmonella choleraesuis was constructed through plasmid PYA3493contained asdgene entered into ΔcrpΔasdC78-1mutant. The KM mouse lethal showed that thevirulence of the ΔcrpΔasdC78-1mutant was lower780times or1.5times than C78-1or C500, and this system can be used to carry gene stably as a carrier of live vaccine.3. Construction and identification for attenuated Salmonella choleraesuis recombinedwith ORF5of PRRSVThe dORF5gene without N-signal peptide was amplified by RT-PCR frompositive sample of PRRS, then cloned into plasmid of PYA3493. And the recombinebacterium of crp asdC78-1(PYA-dORF5) was abtained successfully when PYA-dORF5entered into crp asdC78-1with a method of electrotransformation. And thegene of dORF5can be transmited stablely in the liquid nutrient medium of LuriaBertani after60generation; Aslo, the identification for successful expressing ofdORF5gene was carried out by SDS-PAGE electrophoresis and Western blot, and theexpression product had a better reactionogenicity, and an establish grounding will beconstructed for live vaccine of PRRS.