摘要
目的通过构建结核分枝杆菌蛋白TB16.3的原核表达载体并表达纯化,对其检测抗体的性能进行评价。方法用PCR法从结核菌H37Rv基因组DNA扩增TB16.3基因,连接到表达载体PET-30a上,在大肠杆菌中表达;组氨酸标签镍柱层析纯化重组蛋白。结果建立ELISA方法检测116份TB患者和96份健康对照者血清中的抗结核菌TB16.3抗体。构建了表达TB16.3的大肠杆菌工程菌,发现目的蛋白主要以可溶形式存在。诱导剂IPTG浓度为1mmol/L,37℃培养5h可获得最大量表达。而在28℃时重组蛋白为可溶形式表达。用重组TB16.3蛋白,ELISA方法检测118份TB患者和96份健康者血清,敏感性、特异性和调整一致率分别为72.9%、86.5%和79.6%。抗TB15.3抗体的敏感性、特异性和调整一致率分别为46.6%、99.0%和76.0%。抗CFP-10的敏感性、特异性和调整一致率分别为75.4%、55.2%和65.7%。结核病患者抗Rv2626C抗体阳性率(44.1%)显著低于其在正常对照的阳性率(75.0%,χ2=20.8,P<0.01)。TB15.3和TB16.3等量包被检测,敏感性、特异性和一致率分别达69.4%、96.9%和83.7%,检测非TB肺部疾病患者,其抗体阳性率仅为9.6%,特异性为90.4%。TB15.3和TB16.3混合后同时包被检测,结合TB15.3单独检测的结果,则敏感性、特异性和一致率分别达82.2%、95.9%和88.9%,一致率为最高。结论目的基因克隆入宿主菌中并表达成功,获得制备上述4种蛋白的最适条件。获得的重组TB16.3、CFP-10和TB15.3蛋白可能成为结核病血清学诊断的抗原或者抗原组合。Rv2626C抗体可能被用于结核患者恢复期的评估。
In order to evaluate the potential of TB16.3 protein in serodiagnosis of TB, Rv2815C gene was cloned and the protein was expressed in Escherichia coli. The gene encoding TB16.3 protein was amplified by PCR from genome of M. tuber culosis H37Rv, and then inserted into expression vector PET30a and expressed fusion proteins TB16.3 in E. coli BL21 （DE3）. The recombinant protein was purified by affinity chromatography. The 118 sera from pulmonary TB patients and 96 sera from controls were detected for anti-TB16.3, anti-TB15. 3, anti-CFP-10 and Rv2626C by ELISA. Results showed that the target protein was expressed in E. coli after induction with IPTG. The solubility analysis showed that TB16.3 existed as soluble pro tein. Soluble TB16.3 was obtained at 28 ℃ culture from E. coli with expressing vector culture filtrate. Maximum output was achieved at the culture time of 5 hours at 37 ℃ with 1 mmol/L IPTG induction. A total of 118 sera from pulmonary TB patients and 96 sera from the normal controls were detected for Ab to TB16.3 by EI.ISA and results showed the sensitivity, specificity and adjusted concordance rate of anti-TB16.3 were 72.9%, 86.5 % and 79.6%, respectively. The sensitivity, specificity and adjusted concordance rate of anti TB15.3 were 46.6%, 99.0% and 76.0% respectively. The sensitivity, specificity and adjusted concordance rate of anti-CFP-10 were 75. 4Yo, 55. 2% and 65.7%, respectively. The anti-TB16.3 positive rate was only 9. 6% among patients of the nomTB pulmonary diseases, which indicated the specificity of TB16.3 antibody was 90.4%. The an tibody to MTB Rv2626C positive rate of TB patients （44. 1%）was significantly lower than that of the controls （75.0%, χ2 =20.8, P〈0.01）. Coated with TB16.3 and TB15.3 equally to detect the both antibodies, the sensitivity, specificity and agreement rate were 69.4 %, 96.9% and 83.7%, respectively. The highest concordance rate was achieved using TB16.3 and TB15.3 as capture combined with TB15.3 for detection along. The sensitivity, specificity and concordance rate were 82.2%, 95.8M and 88.9%, respectively. In conclusion, the gene Rv2815c was obtained and the antigen was expressed in E. coli BL21. The TB16.3, TB15.3 and CFP-10 proteins are potential candidates for the early detection of MTB infection.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2013年第8期791-796,共6页
Chinese Journal of Zoonoses
基金
海南省2008年度重点科技项目(No.080209)资助~~
