肺炎支原体P1重组蛋白的提取纯化及应用研究

Extraction-Purification and Application of P1 Recombinant Protein Antigen of Mycoplama pneumonia

提取纯化肺炎支原体(Mycoplasma pneumoniae,Mp)P1重组蛋白,建立酶联免疫吸附实验(ELISA)的方法,协助临床肺炎支原体感染的诊断。以GST融合蛋白层析柱提取、纯化Mp P1重组蛋白做抗原,以全肺炎支原体菌体成分做抗原对照,建立间接ELISA实验方法,检测40份正常献血者血清标本和51份疑似MP感染临床血清标本的IgG抗体。重组蛋白经SDS-PAGE可见诱导表达的样品在分子量大约59 ku处有明显条带,经Western blotting可与肺炎支原体免疫血清发生反应。ELISA实验检测51份临床标本,由P1重组蛋白抗原检测阳性31份,阳性率为60.78%。Mp检测阳性20份,阳性率为39.22%。实验精确度检测阳性混合血清的变异系数(CV值)为5.40%,阴性混合血清变异系数为1.10%。用Mp P1重组蛋白抗原建立的ELISA检测方法,其敏感性高于全肺炎支原体抗原,可用于临床肺炎支原体感染的诊断。

In order to help clinical diagnosis of Mycoplasma pneumonia （Mp） infection, P1 recombinant protein was extracted and purified to establish its ELISA experimental method. The P1 recombinant protein extracted and purified with B-PER GST Fusion Protein purification Kit was used as an antigen, and thallus component of full Mp was used as an antigen control to establish indirect ELISA experimental method to test and determine IgG antibody of serum sam- ples from 40 normal blood donors and 51 clinical serum of partly certain and partly uncertain Mp infector. The results showed that the recombinant protein expressed by SDS-PAGE visible induction had clear band at about 59 ku, and could react with immune serum of Mp through Western blotting. 31 out of 51 were detected positive using P1 recombinant protein with positive rate at 60.78% , and 20 serum were detected positive using Mp with positive rate at 39.22%. The experiment accuracy test showed that the coefficient of variation （CV） of the positive mixed serum was 5.04% , and the CV of negative mixed serum was 1.10%. Therefore, the sensitivity of ELISA testing method established by Mp P1 recombinant protein antigen was higher than full Mp antigen, and can be used for clinical diagnosis of Mp infection.