犬疥蟎副肌球蛋白抗血清的制备及临床应用

The preparation and clinical application of canin Sarcoptes scabiei anti-paramyosin serum

目的:利用现代分子生物学方法,建立一种准确率更高的犬疥蟎病的诊断方法,为犬疥蟎病的治疗提供诊断基础。方法:收集犬疥蟎,抽提总RNA并将其反转录为cDNA;以该cDNA为模板,参考其它哺乳动物副肌球蛋白的基因序列特征,设计引物扩增含有多个抗原表位的犬副肌球蛋白的部分cDNA序列;构建原核表达载体,表达及纯化目的蛋白片段;以纯化的目的蛋白片段为抗原,免疫BALB/c小鼠制备抗血清;以制备的抗血清为一抗,通过间接ELISA法测定患病犬痂皮组织中疥蟎副肌球蛋白片段的存在,进而判断是否有疥蟎感染。结果:成功构建了副肌球蛋白的原核表达载体,命名为pET28a-Para-202;通过诱导、纯化获得了重组蛋白;该重组蛋白抗原免疫小鼠后获得高效价的抗血清;以该抗血清为工具,通过间接ELISA法极大提高了犬疥蟎感染的诊断率,为该病治疗提供了重要的参考依据。

Obective： the research was aimed to establish one new method for diagnosing canin Sarcoptes scabiei more ef- ficiently by using methods in modern molecular biology. Method： canin Sarcoptes scabiei was collected and its total RNA was extracted and reverse transcribed into cDNA. Specific primers were designed to amplify partical paramyosin cDNA products with several epitopes from the total cDNA. The partial paramyosin cDNA was then subcloned into pET28a（＋） vector and transformed into competent Escherichia coIi BL21 （DE3） for expression. Recombinant protein was purified,renaturated,and used as antigen for anti-serum production. With the anti-serum, indirect ELISA was used to detected the existence of native paramyosin in sicken crusta. Result： recombinant plasmid pET28a-Para-202 was constructed and the purified recombinant paramyosin was available. The protein had relative high immunogenicity and the anti-paramyosin serum with high IgG levels was acquired. With the anti-paramyosin serum, the detection rate of canin Sarcoptes scabiei was improved apparently.