摘要
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。
Objectives To construct a vector for prokaryotic expression of the human procalcitonin （PCT） gene, to ex- press it in Escherichia coli, to prepare anti human PCT monoclonal antibodies, and to establish and evaluate a chemilumi- nescence immunoassay for efficient detection of human PCT. Methods In accordance with the sequence of the PCT gene published by the NCBI, the gene sequence was optimized based on the codon preference of E. coll. Restriction sites and protective bases were added to the gene sequence, and then the entire gene was synthesized. A prokaryotic expression vector was constructed and the DNA sequence of PCT was cloned into pET32a and transformed into E. coli BL21（DE3）. Expression of protein by the recombinant plasmid pET32a-PCT was induced with 0.4, mmol/L of IPTG for 6 h. Expres sion of the recombinant protein was analyzed with Western blot analysis. The recombinant protein was purified with Ni affinity chromatography. BALB/c mice were immunized with the recombinant PCT protein. After the serum titer was higher than 1：64000, spleen cells of immunized mice were fused with SP2/0 cells. Positive hybridomas were obtained u- sing indirect ELISA. Positive monoclonal cells were selected using limited dilution. Positive monoclonal cells were injec- ted into the abdominal cavity of mice to produce monoclonal antibodies （mABs） in ascitie fluid. The mAbs were purified with Protein A/G, and the purified mAbs were identified with SDS-PAGE. The specificity and sensitivity of the mAbs were determined using indirect ELISA. The monoclonal antibodies were labeled with HRP and the paired monoclonal anti-bodies were screened using checkerboard titration. Based on the paired antibodies that were identified, a serological sys tern was created to test 150 serum samples. Results The recombinant human PCT protein that was expressed had a molecular mass consistent with what was expected （30 ku） according to Western blot analysis. Western blot analysis indi cared that the pET32a-PCT protein was recognized
出处
《中国病原生物学杂志》
CSCD
北大核心
2017年第4期306-310,316,共6页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.J1210026)
江苏省自然科学基金项目(No.BK20161408)
关键词
单克隆抗体
人降钙素原(PCT)
双抗体夹心
定量检测
monoclonal antibody
human procalcitonin （PCT）
double anti-sandwich
quantitative detection
