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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。
Objective To prepare multiple antiserum for detection of MHC class I proteins with different MHC-B haplotypes and to compare the susceptibility of BW / G3 chickens (MHC B2 haplotypes) with stronger Marek’s disease (MD) Strong BW / G7 chickens (B19 haplotype) After inoculation of Marek’s disease virus (MDV), Western blot was used to semiquantitatively analyze the differences in MHC class I protein expression. Methods The 4 ~ 8 exons of the conserved sequence of BF gene were amplified by PCR from the chicken / spleen tissue of BW / G3 strain and cloned into the prokaryotic expression vector pET-30a to construct a recombinant plasmid. The E.coli BL21 (DE3) was transformed into E.coli BL21 (DE3) and induced by IPTG. The recombinant protein was purified by SDS-PAGE and immunized New Zealand rabbits to prepare rabbit antiserum. The high titer rabbit antiserum was used as the primary antibody to detect the expression of MHC class Ⅰ protein in healthy chickens of BW / G3 and BW / G7 strains and the 11th week after challenge with MDV. Western Blot was performed using ImageQuant 5.2 software Variety. Results The recombinant plasmid containing BF gene was expressed in E. coli. The specific rabbit antiserum could detect the MHC class Ⅰ protein of different MHC - B haplotype chicken. The expression of MHC class Ⅰ protein in BW / G3 and BW / G7 chickens infected with MDV was significantly higher than that in BW / G3 (P≤0.01, P≤0.001). Conclusion MHC class I specific polyclonal antibodies against MHC B2, B6 and B19 haplotype chickens were successfully prepared. In MHV class I challenged chicken BW / G3 and BW / G7 sensitive chickens, Protein expression levels are upregulated, and the expression of susceptible chickens is higher than that of the resistant chickens, providing ideas for further revealing the pathogenesis of viral neoplastic diseases.