首页|结核分枝杆菌H37Ra株HspX蛋白的表达条件优化、纯化和鉴定

结核分枝杆菌H37Ra株HspX蛋白的表达条件优化、纯化和鉴定

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以结核分枝杆菌H37Ra菌株基因组DNA为模板,利用PCR技术扩增获得HspX基因,通过DNA无缝克隆技术将其克隆至pET28a质粒中,构建重组表达质粒pET28a-HspX.将pET28a-HspX转化至大肠埃希菌表达菌株BL21(DE3),采用不同温度、IPTG浓度和时间诱导HspX蛋白表达.使用Ni-IDA亲和层析柱纯化目的HspX蛋白,透析去除咪唑,通过Western blot检测HspX抗原特异性.最终确定表达重组蛋白HspX的最佳诱导条件为:诱导温度37℃、IPTG浓度0.2 mmol/L、诱导时间8h.结果表明,获得了高纯度和被特异性识别的可溶性HspX蛋白,为未来HspX蛋白用于结核病诊断试剂和疫苗奠定基础.
Optimization of expression conditions,purification and identification of HspX protein from Mycobacterium tuberculosis H37Ra strain
Using the genomic DNA of Mycobacterium tuberculosis strain H37Ra as a template,the HspX gene was amplified by PCR technology and cloned into the plasmid pET28a by seamless DNA cloning technology,and the recombinant expression plasmid pET28a-Hspx was constructed.The pET28a-HspX was transformed into Escherichia coli expression strain BL21(DE3)and HspX protein expression was induced by different temperatures,IPTG concentrations and times.The target HspX protein was purified by Ni-IDA affinity chromatography,imidazole was removed by dialysis,and the antigen specificity of HspX was detected by Western blot.Finally,the optimal induction conditions for the expression of recombinant protein HspX were determined as follows:induction temperature 37℃,IPTG concentration 0.2 mmol/L,induction time 8 h.In conclusion,this study obtained soluble HspX protein with high purity and specific recognition,laying a foundation for the future use of HspX protein in TB diagnostic reagents and vaccines.

Mycobacterium tuberculosisHspXprokaryotic expressionpurificationconditions optimization

魏婧、郑如明、李康生、李柏青、许涛、汪洪涛

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蚌埠医学院检验医学院检验医学实验中心,蚌埠 233030

蚌埠医学院慢性疾病免疫学基础与临床安徽省重点实验室,蚌埠 233030

蚌埠医学院检验医学院免疫学教研室,蚌埠 233030

蚌埠医学院检验医学院临床检验诊断学教研室,蚌埠 233030

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结核分枝杆菌 HspX 原核表达 纯化 条件优化

安徽省自然科学基金安徽省自然科学基金慢性疾病免疫学基础与临床安徽省重点实验室开放课题基金呼吸系病临床基础安徽省重点实验室开放课题基金蚌埠医学院512人才培育计划

1908085MH2522008085QH405KLICD-2002-Z3HX2022-Z02by51201309

2023

10.13488/j.smhx.20220650

生命的化学
中国生物化学与分子生物学会

生命的化学

CSTPCD
影响因子：0.404
ISSN：1000-1336
年,卷(期)：2023.43(2)
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