安氏隐孢子虫肌动蛋白部分编码基因的表达及免疫保护性

以筛选安氏隐孢子虫T7噬菌体展示文库获得的肌动蛋白（CA42）部分编码基因的序列设计特异性引物，扩增目的基因CA42，构建重组表达载体pET-28a-CA42，通过大肠杆菌BL21的诱导表达，产物经SDS-PAGE和Western-blotting鉴定。然后纯化重组蛋白，免疫BALB／c进行体液和细胞免疫水平的检测，并观察重组蛋白对微小隐孢子虫的交叉保护性效果。结果显示，成功构建重组原核表达质粒pET-28a-CA42，Western-blotting显示重组蛋白约为19ku，能被安氏隐孢子虫免疫小鼠的多克隆抗体识别。重组蛋白免疫BALB／c小鼠的抗体水平差异显著（P〈0．05），CD4^＋T淋巴细胞数差异均显著，CD4^4／CD8^＋T淋巴细胞数的值与佐剂对照组和空白对照组相比差异均显著（P〈0．05）。各组小鼠经接种微小隐孢子虫卵囊后，试验组与各对照组相比卵囊减少率为32．2％，差异均显著（P〈0．05）。结果表明，成功表达了重组安氏隐孢子虫肌动蛋白，纯化的重组蛋白具有一定的免疫原性和交叉保护性。

Expression and immunoprotective effect of the actin of Cryptosporidium andersoni

To express the partial enconding sequence of actin of Cryptosporidium andersoni in E.coli and indentify the immunoprotective of recombinant protein.primers was designed for actin gene that was screened from the T7 phage display library of Cryptosporidium andersoni and the actin gene CA42 was amplified by PCR.Recombinant prokaryotic expression plasmid pET28a-CA42 was constructed.The recombinant prokaryotic expression plasmids were transformed into competent E.coli DE3.The expression was induced by IPTG and identified by SDS-PAGE and Western-blotting.Then the protein was purified.Humoral immunity and cell immunity was detected by ELISA and flow cytometry in immunized BALB/c mice.then the mice were inoculated with 1×106 Cryptosporidium parvum oocysts to observe the cross-protection.Western Blotting show that the size of the recombinant protein are about 19ku and the protein can be identified by polyclonal antibody from immunized mice of C.andersoni.ELISA test showed the immune serum antibody titer significantly increase compared with the control group(P<0.05).CD4+ T lymphocytes were significantly increase compared with the control group,CD4+/ CD8+ T lymphocytes were significantly increase compared with the control group(P<0.05).The results of mice inoculated with 1×106 Cryptosporidium parvum oocysts showed that the oocysts number of grams in the experimental group fecal were decreased compared with the control group.Reduction rate of CA42 group was 32.2%(P<0.05).The genes encoding partial actin of C.andersoni has been successfully expressed,The results showed that the recombinant protein has immunogenicity and cross-protection.