中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2008年
6期
492-494
,共3页
周伯平%刘威龙%谢靖婧%陈心春%徐六妹%谭艳%刘映霞%杨桂林
週伯平%劉威龍%謝靖婧%陳心春%徐六妹%譚豔%劉映霞%楊桂林
주백평%류위룡%사정청%진심춘%서륙매%담염%류영하%양계림
肠道病毒71型,人%重组蛋白%血清学诊断%酶联免疫吸附测定
腸道病毒71型,人%重組蛋白%血清學診斷%酶聯免疫吸附測定
장도병독71형,인%중조단백%혈청학진단%매련면역흡부측정
Enterovirus type 71,human%Recombinant protein%Serological diagnosis%Engyme-linked immunosorbent assay
目的 获得纯化的具有免疫活性的肠道病毒71型VP1蛋白,建立EV71感染早期、快速和准确的ELISA血清学诊断方法.方法 通过PCR方法扩增出VP1基因,定向克隆到原核表达载体pET-21b(+),阳性质粒转化入E.coli B121(DE3)感受态细胞经IPTG诱导,SDS-PAGE电泳和蛋白免疫印迹分析目的 蛋白的表达水平.纯化的VPI蛋白用作包被抗原,建立手足口病(HFMD)患者抗-EV71-IgM和IgG的血清学诊断方法.结果 成功表达和纯化了VP1重组蛋白,所表达的蛋白能被EV71型手足口病患者血清所识别.调查发现,与正常人和EV71阴性手足口病患儿比较,EV71阳性手足口病血清中抗-EV71-IgM和IgG中A值显著升高.差异具有统计学意义(P<0.05).与RT-PCR结果比较,发现该方法IgM的诊断敏感性和特异性分别为73%和77%;该IgG诊断方法的敏感性和特异性分别为82%和83%.完成该试验仪需4 h.结论 利用pET原核表达系统成功克隆、表达和纯化了肠道病毒71型重组外壳蛋白VPI,且具有良好的抗原性.该抗原可用于研制EV71血清学诊断试剂盒.
目的 穫得純化的具有免疫活性的腸道病毒71型VP1蛋白,建立EV71感染早期、快速和準確的ELISA血清學診斷方法.方法 通過PCR方法擴增齣VP1基因,定嚮剋隆到原覈錶達載體pET-21b(+),暘性質粒轉化入E.coli B121(DE3)感受態細胞經IPTG誘導,SDS-PAGE電泳和蛋白免疫印跡分析目的 蛋白的錶達水平.純化的VPI蛋白用作包被抗原,建立手足口病(HFMD)患者抗-EV71-IgM和IgG的血清學診斷方法.結果 成功錶達和純化瞭VP1重組蛋白,所錶達的蛋白能被EV71型手足口病患者血清所識彆.調查髮現,與正常人和EV71陰性手足口病患兒比較,EV71暘性手足口病血清中抗-EV71-IgM和IgG中A值顯著升高.差異具有統計學意義(P<0.05).與RT-PCR結果比較,髮現該方法IgM的診斷敏感性和特異性分彆為73%和77%;該IgG診斷方法的敏感性和特異性分彆為82%和83%.完成該試驗儀需4 h.結論 利用pET原覈錶達繫統成功剋隆、錶達和純化瞭腸道病毒71型重組外殼蛋白VPI,且具有良好的抗原性.該抗原可用于研製EV71血清學診斷試劑盒.
목적 획득순화적구유면역활성적장도병독71형VP1단백,건립EV71감염조기、쾌속화준학적ELISA혈청학진단방법.방법 통과PCR방법확증출VP1기인,정향극륭도원핵표체재체pET-21b(+),양성질립전화입E.coli B121(DE3)감수태세포경IPTG유도,SDS-PAGE전영화단백면역인적분석목적 단백적표체수평.순화적VPI단백용작포피항원,건립수족구병(HFMD)환자항-EV71-IgM화IgG적혈청학진단방법.결과 성공표체화순화료VP1중조단백,소표체적단백능피EV71형수족구병환자혈청소식별.조사발현,여정상인화EV71음성수족구병환인비교,EV71양성수족구병혈청중항-EV71-IgM화IgG중A치현저승고.차이구유통계학의의(P<0.05).여RT-PCR결과비교,발현해방법IgM적진단민감성화특이성분별위73%화77%;해IgG진단방법적민감성화특이성분별위82%화83%.완성해시험의수4 h.결론 이용pET원핵표체계통성공극륭、표체화순화료장도병독71형중조외각단백VPI,차구유량호적항원성.해항원가용우연제EV71혈청학진단시제합.
Objective To obtain a recombinant purified Enterovims 71 VP1 protein and establishment of an early,rapid and accurate serolngieal ELISA (enzyme-linked immunesorbent assay)for detection of EV71 infection. Methods VPI gene was amplified by PCR and cloned into pET-21b ( + ) vector,the positive recombinant plasmid were transformed into E. coil BI21 ( DE3 ),and was induced with IPTG,the recombinant protein by SDS-PAGE and Western Blot assays. Finally,the recombinant purified VP1 protein was used as a coated antigen for detection of serum anti-lgM and lgG against EV71 by ELISA. Results The purified VP1 was obtained,and it can be recognized by sera of patients with EV71 infection associated with hand-foot-mouth disease. The A values of anti-EV71 lgM and IgG were significantly elevated as compared to healthy objects and HFMD patients without EV71 infection ( P < 0.05) .The sensitivity and specificity of IgM to EV71 were 73% and 77% compared with the RT-PCR results,respectively; and those of IgG being 82% and 83%,respectively. Conclusions The recombinant protein VP1 was produced and purified,and it was proved to have a good antigenicity and eould be used to develop a serological diagnosis kit for EV71 infection in the future.
