CAJ | 학술논문

目的表达A组轮状病毒疫苗株LLR的VP8*蛋白,为深入研究LLR的功能特性奠定基础.方法从兰州生物制品所生产的A组轮状病毒疫苗中提取病毒RNA为模板,经过RT-PCR反应获得轮状病毒LLR株VP8*基因的cDNA片段,将其克隆到pGEX-4T1表达载体中构建重组质粒pGEX-4T1-VP8*,测序正确后转化大肠埃希菌BL21(DE3),通过SDS-PAGE电泳、免疫印迹方法检测IPTG诱导表达后的产物,用Glutathione Sepharose 4B纯化目的蛋白.结果 SDS-PAGE电泳可见大小约为52 000的融合蛋白,Western-Blot显示该蛋白能与GST标签抗体结合.结论成功构建了包含LLR株VP8*序列的重组表达载体pGEX-4T1-VP8*,获得了可溶性GST-VP8*重组蛋白.
목적 표체A조륜상병독역묘주LLR적VP8*단백,위심입연구LLR적공능특성전정기출.방법 종란주생물제품소생산적A조륜상병독역묘중제취병독RNA위모판,경과RT-PCR반응획득륜상병독LLR주VP8*기인적cDNA편단,장기극륭도pGEX-4T1표체재체중구건중조질립pGEX-4T1-VP8*,측서정학후전화대장애희균BL21(DE3),통과SDS-PAGE전영、면역인적방법검측IPTG유도표체후적산물,용Glutathione Sepharose 4B순화목적단백.결과 SDS-PAGE전영가견대소약위52 000적융합단백,Western-Blot현시해단백능여GST표첨항체결합.결론 성공구건료포함LLR주VP8*서렬적중조표체재체pGEX-4T1-VP8*,획득료가용성GST-VP8*중조단백.
Objective To express the VP8 * protein of the rotavirus vaccine strain LLR in the E.coli with the pGEX4T-1 vector,which is important for the further research of the VP8 * protein functions.Methods The LLR VP8 * gene was obtained by virus RNA extraction and RT-PCR.Then it was cloned in the expression vectors pGEX4T-1.The recombinant plasmid pGEX4T-1-VP8 * was transformed to the E.coli BL21.Then the VP8 *-GST fusion protein was expressed and purified by the affinity chromatograph.The protein of interest was validated by SDS-PAGE and Western Blot.Results The molecular weight of the VP8 *-GST fusion protein was about 52 000 according to the SDS-PAGE.The bands of both 52 000 and 26 000 were shown in the Western Blot with the antibody against GST.Conclusions The LLR VP8 * gene was obtained and cloned to the pGEX4T-1 vector.Moreover,the solvable VP8 *-GST fusion protein was successfully expressed and purified.