鸡MHC Ⅰ类分子的多抗制备及其在不同MD抗性MHC单倍型鸡脾脏中的差异表达

Preparation of specific polyclonal antibody and differential expression of MHC class Ⅰ proin in spleens of MD resistant and susceptible chicken lines

目的制备能检测不同MHC-B单倍型MHC I类蛋白的多抗血清,并对马立克氏病(MD)抗性较强的BW/G3系鸡群(MHC B2单倍型)和敏感性较强的BW/G7系鸡群(B19单倍型)接种马立克氏病毒(MDV)后,利用Western blot技术半定量分析MHC I类蛋白表达量的差异。方法从BW/G3系鸡脾组织cDNA中PCR扩增BF基因的保守序列第4～8外显子,克隆入原核表达载体pET-30a,构建重组质粒。转化大肠杆菌BL21(DE3),经IPTG诱导获得表达,经SDS-PAGE初步纯化,免疫新西兰兔,制备兔抗血清。以高效价的兔抗血清为一抗,对BW/G3和BW/G7系健康鸡和MDV攻毒后11周的脾组织进行Western blot,利用ImageQuant 5.2软件半定量分析其中MHC Ⅰ类蛋白含量的变化。结果含BF部分基因的重组质粒在大肠杆菌中表达,其特异性兔抗血清能检测到不同MHC-B单倍型鸡的MHC Ⅰ类蛋白。BW/G3和BW/G7系鸡群感染MDV后,MHC Ⅰ类蛋白含量全部上调,正常组和攻毒组中BW/G7系的表达量均极显著高于BW/G3系(P≤0.01,P≤0.001)。结论成功制备了能检测MHC B2、B6和B19单倍型鸡的MHC I类蛋白特异性多抗血清;MDV攻毒后期,抗性鸡BW/G3和敏感鸡BW/G7脾组织中MHC I类蛋白的表达量都发生上调,且易感鸡中的表达量高于抗性鸡,为进一步揭示病毒性肿瘤病的致病机理提供了思路。

To semi-quantitatively analyze differential expression of MHC class Ⅰ protein in MDV-infected and control chickens of BW/G3 and BW/G7 lines,we cloned and expressed BF（exon4-8） gene encoding MHC class Ⅰ α chain protein.The target fragment was successfully amplified and then cloned into prokaryotic expression plasmid pET-30a.After identification by restriction endonuclease digestion and direct DNA sequencing,the recombinant plasmid pET-BF was transformed into E.coli BL21.After induction by IPTG,the isolated protein was analyzed with SDS-PAGE.The rabbit serum was harvested after 5 times of immunization with the primary purified recombinant protein,and then analyzed by Western blotting.The differential expression of MHC class I protein in MDV-infected and control chickens of BW/G3 and BW/G7 lines were semi-quantitatively analyzed using the ImageQuant 5.2 software.As showed by Western blotting,the polyclonal antibody of the rabbit serum could detect MHC class I protein of different MHC-B haplotypes.The expression of MHC class I protein was increased in MDV-infected chickens of BW/G3 and BW/G7 lines.The expression of MHC class I protein of BW/G7 chickens was higher than that of infected and control BW/G3 chickens.All results indicate that the polyclonal antibody of the rabbit serum is prepared successfully,the expression of MHC class Ⅰ genes of the resistant and susceptible chickens are up-regulated during the late stage of the infection,and the expression of MHC class I genes of susceptible chickens was higher than that of resistant chickens,which would provide clues for exploring the pathogenic mechanism of virulence oncosis.