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目的重组原核表达人降钙素原(PCT)蛋白,制备抗人PCT单克隆抗体,建立高效检测PCT化学发光免疫分析方法。方法根据NCBI提供的PCT基因序列,按照大肠埃希菌偏爱密码子优化后进行全基因合成,通过BamHⅠ和XhoⅠ双酶切将其构建到pET32a载体上;将pET32a-PCT质粒转化至大肠埃希菌BL21(DE3),用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,采用Western blot法分析重组蛋白表达情况;用镍亲和纯化柱纯化重组蛋白,以此为抗原免疫BALB/c小鼠。取免疫小鼠脾细胞与小鼠骨髓瘤SP2/0细胞融合,采用间接ELISA法筛选阳性杂交瘤细胞,有限稀释法进行克隆化后再用间接ELISA法筛选阳性单克隆细胞;将阳性单克隆细胞接种于小鼠腹腔内,制备腹水单抗,经Protein A/G纯化后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,采用间接ELISA方法对抗体的特异性、灵敏度以及亲和力进行鉴定;用NaIO4氧化HRP法标记单克隆抗体,通过棋盘法筛选配对单克隆抗体,以筛选的配对抗体为捕捉抗体,建立双抗夹心化学发光免疫分析的血清学检测体系,检测临床150例血清标本,其中PCT升高(>0.05ng/ml)120例,PCT阴性(<0.05ng/ml)30例。结果共筛选出6株抗人PCT蛋白的单克隆细胞,制备的腹水效价均大于4×10~(-6);通过棋盘法筛选得到2对能够进行双抗夹心配对的单抗,其中1对亲和力较高,其亲和力常数分别为2.39×10~8 L/mol和2.91×10~8 L/mol。双抗夹心化学发光免疫分析方法检测线性范围为0.06~8ng/ml,其中阳性检出率为96.6%,阴性符合率为100%,与临床检测数据相比,相关系数R~2=0.9737。结论建立的双抗夹心ELISA方法灵敏度高、特异性强、稳定可靠,可用于细菌、寄生虫等病原体感染患者的PCT血清学定量检测。
Objective To construct prokaryotic expression of human procalcitonin (PCT) protein and prepare anti-human PCT monoclonal antibody (McAb), and to establish a high-performance chemiluminescent immunoassay for detection of PCT. Methods According to the PCT gene sequence provided by NCBI, the whole gene was synthesized according to the preferred codon usage of Escherichia coli, and then was digested with BamHⅠ and XhoⅠ to construct pET32a vector. The plasmid pET32a-PCT was transformed into Escherichia coli BL21 (DE3) was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression of recombinant protein was analyzed by Western blot. The recombinant protein was purified by nickel affinity purification column, BALB / c mice. Immunized mouse spleen cells were fused with mouse myeloma SP2 / 0 cells. Positive hybridoma cells were screened by indirect ELISA, cloned by limiting dilution method, and then positive monoclonal cells were screened by indirect ELISA. Positive monoclonal cells Were inoculated intraperitoneally into mice to prepare monoclonal antibody of ascites. The purified protein was purified by Protein A / G and identified by SDS-PAGE. The specificity, Sensitivity and affinity. The monoclonal antibodies were labeled with NaIO4 oxidized HRP method, and the matched monoclonal antibodies were screened by checkerboard method. The screening matched antibodies were used as the capture antibody to establish the double antibody sandwich chemiluminescence immunoassay serological test system, 150 cases of serum samples, including PCT increased (> 0.05ng / ml) in 120 cases, PCT negative (<0.05ng / ml) in 30 cases. Results Six monoclonal anti-human PCT clones were screened and the ascites titer was higher than 4 × 10 -6. Two pairs of McAbs were obtained by the checkerboard method, 1 had higher affinity and their affinity constants were 2.39 × 10 ~ 8 L / mol and 2.91 × 10 ~ 8 L / mol, respectively. The linear range of double antibody sandwich chemiluminescence immunoassay was 0.06 ~ 8ng / ml. The positive detection rate was 96.6% and the negative coincidence rate was 100%. The correlation coefficient was R ~ 2 = 0.9737 compared with the clinical detection data. Conclusion The established double-antibody sandwich ELISA method has the advantages of high sensitivity, strong specificity, stable and reliable, and can be used for the quantitative detection of PCT serology in patients with bacterial, parasitic and other pathogen infections.