CAJ | 학술논문

目的比较不同tPA信号肽突变体对目的蛋白表达和分泌水平的影响,优化并筛选通用性外源信号肽序列.方法通过定点突变技术将人类组织型纤溶酶原激活物(tPA)的信号肽第22位氨基酸由脯氨酸(P)突变为丙氨酸(tPA22P/A)或甘氨酸(tPA22P/G),并将突变前后的3种信号肽序列分别插入HIV-1 p24基因的N末端,构建不同的p24蛋白表达载体,这些重组表达载体体外瞬时转染人胚肾HEK293T细胞,转染72 h后,通过SDS-PAGE与western blot检测并比较各组培养上清和细胞中p24蛋白的表达水平.结果成功构建了3个p24蛋白的真核表达载体P24T.tPA、P24T.tPA22P/A和P24T.tPA22P/G,经序列测定证实基因序列和方向与预期相符;重组载体转染293T细胞72 h后均检测到分泌性p24的表达.与P24T.tPA组相比,P24T.tPA22P/A转染组培养上清中和细胞内p24蛋白表达水平分别提高62%和29%,P24T.tPA22P/G转染组分别提高8%和6%.结论 tPA信号肽22位由脯氨酸突变为丙氨酸或甘氨酸,这就提高了目的蛋白的表达和分泌水平,为优化外源蛋白在哺乳细胞中的表达和分泌提供了实验基础.
목적 비교불동tPA신호태돌변체대목적단백표체화분비수평적영향,우화병사선통용성외원신호태서렬.방법 통과정점돌변기술장인류조직형섬용매원격활물(tPA)적신호태제22위안기산유포안산(P)돌변위병안산(tPA22P/A)혹감안산(tPA22P/G),병장돌변전후적3충신호태서렬분별삽입HIV-1 p24기인적N말단,구건불동적p24단백표체재체,저사중조표체재체체외순시전염인배신HEK293T세포,전염72 h후,통과SDS-PAGE여western blot검측병비교각조배양상청화세포중p24단백적표체수평.결과 성공구건료3개p24단백적진핵표체재체P24T.tPA、P24T.tPA22P/A화P24T.tPA22P/G,경서렬측정증실기인서렬화방향여예기상부;중조재체전염293T세포72 h후균검측도분비성p24적표체.여P24T.tPA조상비,P24T.tPA22P/A전염조배양상청중화세포내p24단백표체수평분별제고62%화29%,P24T.tPA22P/G전염조분별제고8%화6%.결론 tPA신호태22위유포안산돌변위병안산혹감안산,저취제고료목적단백적표체화분비수평,위우화외원단백재포유세포중적표체화분비제공료실험기출.
Objective To optimize the tissue plasminogen activator (tPA) signal peptide (tPA-SP) as a universal heterologous signal peptide for the secretory expression of target proteins. Methods Two tPA-SP mutants, tPA22P/A and tPA22P/G, were constructed by substituting the amino acid proline at 22 position of tPA-SP to alanine (A) or glycine (G), respectively. Then, three HIV-1 p24 expression vectors, P24T. tPA,P24T. tPA22P/A and P24T. tPA22P/G were constructed by introducing tPA-SP, tPA22P/A or tPA22P/G into the N terminus of p24 gene. HEK293T cells were transfected by these recombinant vectors. 72h post-transfection, the p24 protein in the supernatant and cell lysate of each transfectant was examined by SDS-PAGE and western blot. Results Three p24 eukaryotic expression vectors P24T. tPA, P24T. tPA22P/A and P24T.tPA22P/G were successfully constructed. The three constructs gave secretory expression of HIV-1 p24 protein.The expression levels of p24 protein driven by P24T. tPA22P/A and P24T. tPA22P/G increased by 62% and 8% in the transferred supernatants, respectively, comparing with that driven by P24T. tPA; and the expressionlevels increased by 29% and 6% in the transferred cell lysates, respectively. Conclusions The amino acid substitution of tPA-SP 22P with alanine or glycine enhanced the expression and secretory levels of the target protein. This work provides an experimental foundation for optimizing the expression and secretion of heterologous proteins in mammalian cells.