The Pathology Observation Of MD Chickens And Quantitative Detection Of Chprnp Genes In Different Diseased Lesions

【Objective】In order to understand the prevalence state of Marek’s disease in the region and changes invirulence and histopathological changes, the diagnosis and pathology research of MDV in kangle county,Gansu Province farms suspected MD of green feet chickens were studied; the process of real-time PCR forexpression of PRNP was established to analysis ChPRNP dynamic expression pattern in disease chickens ofMD different lesions and to reveal MD involved PrPCin the development of neoplastic disease and to providea scientific basis for the human to quickly overcome cancer theoretical basis.【Methods】The green feet chicken suspected MD in kangle county, Gansu Province farms as test materials,disease and healthy chicken feathers and blood and marrow, heart, liver, spleen, lung, kidney, brain, spinal cordand other tissue samples were random collected,(1) primers was designed according to the NCBI has beenincluded MDV meq gene to analyze variation feature MDV strains test method by PCR, cloning, sequenceanalysising, etc;(2) dissectived MD chickens, diseased of tissues were collected in4%paraformaldehyde fixedparaffin-embedded sections, MD chickens histopathological were strained and analysied by HE;(3) total RNAwas extracted and transcribed into cDNA from different lesions, no lesions of MD and normal tissue samples,ChPRNP SYBR Green I double standard curve method of fluorescence quantitative PCR detection method anddouble standard curve of ChPRNP gene,-actin gene were established, real-time PCR amplification conditionswere optimized, the result was into a standard curve to analysis the expression level changes of ChPRNP genesof MD chicken in different tissues,statistical analysis of differential expression in different tissues ChPRNP.【Results】(1) AGP test of feather pulp was detected positive rate over99.9%, in PCR for meq gene amplifiedto consistent with the predicted804bp fragment size, the positive rate was100%; the pandemic strains withreference strain meq gene nucleotide homology of98.3%and amino acid homology of96.6%were revealedby cloning meq gene and analysising sequence, the pandemic strains with the0095, GXY2of china in recentyears nucleotide homology over99.3%and amino acid homology of98.8%, higher than the domestictraditional strains RB1B,648a, MD5, MD11, vaccine strains CVI988, pandemic strains were96.6%-100.0%, amino acid homology of98.4%-100.0%; according to the characteristics of meq genes nucleotidesequence of GXY2,0095attenuated in the319th was transform from T to C and in the502th was transformfrom C to G, two changes were specific from pandemic strains in nearly years, other reference strains forcomparison have been not changed; the pandemic strains with domestic reference strains GXY2,0095were ona branch and with the foreign strains RB1B, MD5, MD11,648A and domestic strains N were differentbranches. phylogenetic analysis showed that mutant strains kinship closed with domestic reference strainsGXY2,0095and had a distant relatives with the foreign reference strains;(2) pathological autopsy of MDchickens obwerved,swelling, bleeding, large and small tumor nodules of pathological examination of tissuesand organs were seen clearly; histological examination showed nerve tissue infiltrated lymphocyte-like cell proliferation and demyelinated and had different degrees of edema and proliferated a small plastic stromal cell,the visceral tissues had diffuse or focal tumor cell invasion in different degree;(3) The real-time fluorescencequantitative PCR of SYBR Green I double standard curve method was optimizated to analysis ChPRNP oftissues in MD chickens;(4) ChPRNP genes were expressed in heart, liver, spleen, lung, kidney, brain andspinal cord of12weeks and16weeks MD chickens; normalization values had significant differences(P<0.05)in each week-old lesions, non-diseased tissues and normal tissues; ChPRNP genes expressiondifferences multiple had differences in diffenent lesions, but the differences multiples had differents, thelesions had than non-diseased tissues, normal tissues in addition to individual organizations.【Conclusion】(1) The AGP and PCR of suspected MD of green feet chicken in kangle county, GansuProvince farms were positive, it was indicated that the farm chickens have been infected with MD;(2) Thepandemic strains and reference strains of meq gene with the evolution correlated geographically and with thedomestic isolates GXY2,0095close genetic relationship;(3) Pathological autopsy shows, but pathologicalchange arising from different organizations, histological observation showed that a large number of tumorcells showed typical lesions;(4) ChPRNP genes SYBR Green I real-time PCR detection method wassuccessfully established to analysis ChPRNP of tissues in MD chickens;(5) ChPRNP genes expression levelsare closely related to MDV infection, in different lesions, non-diseased tissues, normal tissues of sameweek-old had differences in ChPRNP genes expression levels and diseased tissues had than non-diseasedtissues, normal tissues; the same lesions, non-diseased tissues, normal tissues of different week-old haddifferences in ChPRNP genes expression levels.