CAJ | 학술논문

以猪圆环病毒2型(Porcine circovirus type 2，PCV2) WH株基因组DNA为模板，扩增ORF2截短基因，经BamH I/Not I 双酶切处理后与经相同酶切处理的 pET32a (+)原核表达载体连接，获得重组质粒 pET32a-Cap2。将重组质粒转化至 BL21（DE3），经IPTG诱导，对表达产物进行SDS-PAGE和Western blot分析。纯化的重组蛋白免疫小鼠，并对获得的血清进行间接 ELISA检测和中和活性测定。SDS-PAGE分析表明，ORF2截短基因在大肠杆菌中得到表达，蛋白分子质量大小为40 kDa，重组PCV2 Cap蛋白主要以上清的形式存在。Western blot证实重组蛋白能够识别抗PCV2阳性血清。经间接ELISA检测，鼠抗PCV2 Cap血清抗体效价能达到1：25600，间接免疫荧光检测分析表明，鼠抗PCV2 Cap血清能特异性识别PCV2感染细胞中的Cap蛋白。病毒血清中和实验证实，抗PCV2 Cap血清抗体具有中和病毒的活性，中和效价为1：36。猪圆环病毒2型 Cap 蛋白的表达，为进一步研究该蛋白的功能及Cap蛋白亚单位疫苗和检测试剂盒的制备奠定了基础。
이저원배병독2형(Porcine circovirus type 2，PCV2) WH주기인조DNA위모판，확증ORF2절단기인，경BamH I/Not I 쌍매절처리후여경상동매절처리적 pET32a (+)원핵표체재체련접，획득중조질립 pET32a-Cap2。장중조질립전화지 BL21（DE3），경IPTG유도，대표체산물진행SDS-PAGE화Western blot분석。순화적중조단백면역소서，병대획득적혈청진행간접 ELISA검측화중화활성측정。SDS-PAGE분석표명，ORF2절단기인재대장간균중득도표체，단백분자질량대소위40 kDa，중조PCV2 Cap단백주요이상청적형식존재。Western blot증실중조단백능구식별항PCV2양성혈청。경간접ELISA검측，서항PCV2 Cap혈청항체효개능체도1：25600，간접면역형광검측분석표명，서항PCV2 Cap혈청능특이성식별PCV2감염세포중적Cap단백。병독혈청중화실험증실，항PCV2 Cap혈청항체구유중화병독적활성，중화효개위1：36。저원배병독2형 Cap 단백적표체，위진일보연구해단백적공능급Cap단백아단위역묘화검측시제합적제비전정료기출。
The truncated ORF2 of Porcine circovirus type 2(PCV2) WH strain was amplified and cloned into expression vector pET32a (+). The expression vector pET32a-Cap2 was then transformed into BL2 (DE3) for expression with induction of IPTG. The expressed pET32a-Cap products were analyzed in SDS-PAGE and Western blot. The recombinant Cap protein was a soluble protein with molecular weight of 40 kDa. The purified Cap protein was used to immunize mice to prepare antiserum. The resulting murine antiserum had ELISA titer at over 1:25 600 and reacted with PCV2 in infected PK-15 cells in indirect immunofluorescence assay (IFA). The murine antiserum also showed neutralizing titer at 1:36. The availability of the recombinant PCV2 Cap protein provides the basis for development of vaccine and diagnostic reagents.