The Survery Of Marek’s Disease And Establishment Of QRT-PCR For PrP Gene Expression

【Objective】In order to understand the prevalence state of Marek’s disease in the region and the relationshipbetween molecular character of meq gene of prevalent strains and changes in virulence, epidemiologicalinvestigation of Marek’s disease in part of Lanzhou City was studied; prion PRNP was seen as target gene andthe process of real-time PCR for expression of PRNP was the established to analysis PRNP dynamicexpression pattern in CEF with MDV stress infecte and to reveal pathogenic and tumorigenic mechanisms ofPrPC in MDV.【Methods】(1) Agar diffusion test was respectively done using MDV antigen and MDV standard positiveserum. According to internal repeat sequences132bp of MDV in GenBank, primers was designed for PCRtesting. The primers of meq gene were designed for PCR testing to CEF which had pathological changes. Themeq products of PCR was cloned and sequenced. Using software, we made a comparison between thesequence of products and published sequence of meq at home and abroad.(2) According PRNP and-actinsequence in GenBank, each pair of primers was designed. Through optimization of reaction conditions and thesensitivity and specificity test, PRNP SYBR Green I double standard curve method of fluorescencequantitative PCR detection method was established. After appearing pathological changes, collect CEFinfected vaccination MD5, CV1988and the wild strain at different time, total RNA were extracted and reversetranscribed into cDNA and then real-time PCR was carried out. The result was analysised through statistics.【Results】(1) In agar diffusion test,14feather pulp samples were positive and the average infection rate was1.47%. In PCR for132bp,24blood samples were detected and the average infection rate was2.52%. In PCRfor meq gene,7samples were detected, sequence comparison showed that4mutant types of MDV meq genefragment length were804bp. According to the characteristics of nucleotide sequence of attenuated vaccinestains CV1988and814in550~552missing three bases (CAC) and in the213th was transform from G to T,4mutant types were virulent strains. In addition, there were point mutations in some of the reference strain andthe mutant strains in the204th,319th,390th and502th. And there were some corresponding changes in4mutant types’ amino acids. The nucleotide identity among4mutant strains was between99.7%~100.0%. Theamino acid identity among4mutant strains was99.6%-100.0%. Amino acids sequence of meq gene indifferent MDV strains is relatively conservative, and their identity was97.3%~100.0%. The evolutionrelationship of meq gene obviously divided into three categories: the mutant type1,2,3,4, and domesticreference strains G2, GXY2, YL, N,0095were on a large branch and had a distant relatives with the foreignreference strains.(2) The real-time fluorescence quantitative PCR of SYBR Green I double standard curvemethod of PRNP and β-actin was established. Standard plasmid of pMD-PRNP and pMD-β-actin standardcurve respectively were y=-4.218+50.98x, y=-3.575x+46.24, and the sensitivity of them were103.Specificity test showed that amplification products were single and specific. The copy number of PRNP genemRNA reached peaks in3rd d,5th d,6th d with MD5, CV1988and the wild strain stress, respectively. Therewere no significant differences among the PRNP expression quantity of the3stains, but compared with CEFunvaccinated the PRNP expression quantity had significant differences, which pathological changes in3d,5d and6d difference is largest.【Conclusion】(1) In a couple of relatively high correlation between virulence and mutations,there weresimilar changes in meq gene nucleotide sequences between4mutant strains and some of MDV virulent strainpublished at home and abroad in a few mutation sites related to virulence.(2) Amino acids sequence of meqgene in different MDV strains is relatively conservative. The identity nucleotide sequence and homologous amino acid sequence among4mutant strains were respectively high. The mutant strain1,2,3,4, anddomestic reference strains GXY2, YL, N,0095, G2were on a large branch.(3) The infection of MDV had aninfluence to PRNP expression and the influence was started from the level of genes. There was a relationshipbetween expression quantity of PRNP and strain virulence strength. PrPC may play an important role inphysiological function of MDV infection...