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本研究运用犬恶丝虫(Dirofilaria immi-tis)第四期幼虫构建cDNA表达文库,并以第三期幼虫免疫兔制备的多抗进行筛选,从中筛选出一段非常类似于编码钙网蛋白(calreticulin)家族蛋白的cDNA片段。把该片段克隆至pTrcHisB载体,转化到TOP10株大肠杆菌表达融合蛋白。用重组蛋白制备的兔血清来检测虫体是否天然表达钙网蛋白以及运用免疫组化实验进行抗原定位。
In this study, a cDNA expression library was constructed by using the fourth larva of Dirofilaria immiliatus and screened by the polyclonal antibody prepared by the third larvae immunized rabbits. A cDNA fragment encoding the calreticulin ) CDNA fragment of the family of proteins. The fragment was cloned into pTrcHisB vector and transformed into TOP10 E. coli expression fusion protein. Rabbit sera prepared from recombinant protein were used to detect whether the parasites naturally expressed calreticulin and the use of immunohistochemistry for antigen localization.