黏质沙雷菌非特异性核酸内切酶通用型定量检测方法的建立及验证  

Development and verification of a universal quantitative assay for non-specific endonuclease of Serratia marcescens

作　　者：毕华[1] 朱香 卢宁 牛林茹 杨凌 张晓峰 周勇[1] 梁雅丽 BI Hua;ZHU Xiang;LU Ning;NIU Lin-ru;YANG Ling;ZHANG Xiao-feng;ZHOU Yong;LIANG Ya-li(不详;National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院重组药物室,北京100050 [2]山西省生物研究院有限公司,山西太原030000 [3]山西集创生物科技有限公司,山西太原030000 [4]君研生物科技(山西)有限公司,山西太原030000

出　　处：《中国生物制品学杂志》2022年第12期1495-1499,1505,共6页Chinese Journal of Biologicals

基　　金：山西省重点研发计划项目(201903D321087)。

摘　　要：目的建立黏质沙雷菌(Serratia marcescens)Benzonase核酸内切酶(Serratia marcescens Benzonase endonuclease,SMBE)通用型定量检测方法,并进行验证。方法以突变体SMBE(第110位组氨酸突变为丙氨酸)基因BEHA为模板,PCR法扩增BEHA基因,插入至载体pET-20b(+)中,构建重组表达质粒pET-20b(+)-BEHA,经菌落PCR和测序鉴定。重组表达质粒转化E.coli BL21(DE3)pLysS,IPTG诱导表达后进行一步Ni-NTA琼脂糖纯化树脂纯化,纯化产物经15%SDS-PAGE和分光光度计分析,并检测酶活性。用纯化BEHA免疫家兔,以获得的多克隆抗体作为包被抗体,标记HRP的多克隆抗体作为检测抗体,建立SMBE的双抗体夹心ELISA检测法。验证方法的线性范围、准确度、精密性,并确定定量限。采用建立的方法及市售试剂盒分别检测3个厂家生产的SMBE。结果菌落PCR和测序鉴定证明,重组表达质粒pET-20b(+)-BEHA构建正确,第110位氨基酸成功突变为丙氨酸。BEHA的纯度>95%,表达量为3 mg/100 mL,酶活性大幅降低。SMBE标准品在0~20 ng/mL范围内与A_(450)呈良好的线性关系;16、10、0.6 ng/mL的SMBE标准品重复检测3次的偏差分别为-3.06%、-5.40%、3.33%;12和2 ng/mL的SMBE标准品重复10次检测结果CV分别为2.48%和2.91%;定量限为0.2 ng/mL。与市售试剂盒比较,本研究建立的方法对3个厂家生产的SMBE检测结果更接近理论值。结论建立的双抗体夹心ELISA法具有较好的准确度和精密性,该方法可定量检测多种市售SMBE产品,是一种SMBE的通用型定量检测方法。Objective To develop and verify a universal assay for quantitative detection of Serratia marcescens Benzonase endonuclease(SMBE).Methods BEHAgene was amplified by PCR using BEHA gene of SMBE mutant(the 110th histidine mutated to alanine)as template,and then inserted into vector pET-20b(+)to construct the recombinant expression plasmid pET-20b(+)-BEHA,which was identified by colony PCR and sequencing.The recombinant expression plasmid was transformed to E.coli BL21(DE3)pLysS,which was then induced by IPTG and purified by one-step Ni-NTA agarose purified resin.The purified product was analyzed by 15%SDS-PAGE and spectrophotometer,and determined for the enzyme activity,with which rabbits were immunized.Using the obtained polyclonal antibody as carting antibody and the HRP labeled polyclonal antibody as detection antibody,the double antibody sandwich ELISA for SMBE was developed,which was verified for the linearity range,accuracy and precision,and determined for the limit of quantification.SMBE produced by three manufacturers were detected by using the established method and commercially available kits,separately.Results Recombinant expression plasmid pET-20b(+)-BEHA was correctly constructed as identified by colony PCR and sequencing,of which the 110th amino acid was successfully mutated into alanine.The purified BEHA showed a purity of more than 95%,while a greatly reduced enzyme activity with a expression level of 3 mg/100 mL.SMBE standard showed a good linear relationship with A_(450)in the range of 0~20 ng/mL.The deviations of 16,10 and 0.6 ng/mL SMBE standard repeatedly detected for three times were-3.06%,-5.40%and 3.33%,while the CV values of 12 and 2 ng/mL SMBE standard repeatedly detected for10 times were 2.48%and 2.91%,respectively,and the limit of quantification was 0.2 ng/mL.Compared with those of commercially available kits,the results of SMBE produced by three manufacturers determined by the method developed in this study were closer to the theoretical value.Conclusion The double antibody sandwich ELISA

关 键 词：黏质沙雷菌 非特异性核酸内切酶 Benzonase核酸内切酶 双抗体夹心ELISA法 

分 类 号：Q819[生物学—生物工程]