呼吸道合胞病毒F蛋白基因片段真核细胞表达纯化及其ELISA建立的研究

Expression of recombinant respiratory syncytial virus F protein in eukaryotic cells and establishment of ELISA for detecting the protein

目的表达纯化人呼吸道合胞病毒(HRSV)F蛋白基因片段,建立间接夹心酶联免疫吸附分析法(ELISA),为进一步大量表达F基因、构建基因工程疫苗和快速临床检测奠定基础。方法逆转录并扩增F基因中的F1片段,连接到载体中,转染真核细胞,诱导并纯化重组F蛋白。将纯化重组F蛋白免疫小鼠制备抗体血清,并建立间接夹心ELISA。通过100份标本应用"金标法"对所建立的方法进行验证。结果成功获得F基因中F1片段的扩增产物,筛选阳性克隆,得到相对分子质量为45000的大量表达蛋白。确定了间接夹心ELISA的最佳反应条件和工作浓度;鼠抗F1IgG最佳质量浓度为3.2μg/mL,样品最佳反应时间为37℃70 min,酶标兔抗鼠IgG最佳工作浓度为1∶6 000。与"金标法"对比,阳性样品的变异系数为3.2%～8.6%,阴性样品的变异系数为5.1%～8.3%,均小于10.0%,表明该方法有良好的重复性。结论构建的重组真核细胞能够大量表达F蛋白,纯化的F蛋白具有很好的免疫原性,制备的抗体血清用于ELISA能够得到准确的检测结果。

Objective To express human respiratory syncytial virus（HRSV） F protein fragment in eukaryotic system and establish a double antibody sandwich enzyme-linked immunosorbent assay（ELISA） for detecting the protein, so as to lay the foundation for larger scale expression of the F protein, generation of genetic engineering vaccine, and quick detection of the disease in clinic. Methods The cDNA encoding HRSV F protein F1 fragment amplified by RT-PCR was inserted into the expression vector and transformed into COS 27 cells. The recombinant protein was purified after expression and was subsequently used to immunize rat for the generation of antiserum. Double antibody sandwich ELISA was established and verified with 100 specimens in comparison to ＂gold standard＂ method. Results An F gene F1 fragment of 45 000 Daltons was successfully expressed in COS 27 cells. The optimal reaction conditions and working concentration of rat anti-F1 IgG for ELISA were confirmed. It was demonstrated that the optimal concentration of rat anti-F1 IgG was 3.2 Ixg/mL, the best time for sample reaction was 70 minutes at 37 ~C, and the working concentration of enzyme labeled rabbit anti-rat IgG was 1 ： 6 000. Compared with ＂gold standard＂ method, the coefficient of variation was 3.2 % - 8.6 % in the positive sample and 5.1% - 8.3 % in the negative sample. Both of them were less than 10.0 %, indicated that the method had good repeatability. Conclusion It is demonstrated that COS 27 cells could highly express F protein and the purified protein has good immunogenicity. The ELISA method could achieve accurate results using the antibody generated with the purified recombinant protein.