野生型和酶活缺失型人乙酰肝素酶慢病毒表达载体的构建及其验证

Construction and validation of wild type and enzymatic activity dificiency human heparanaselentiviral expression vectors

目的为构建野生型和酶活缺失型人乙酰肝素酶(HPA)的慢病毒表达载体,以探讨HPA的酶活性作用和非酶活性作用。方法根据GV358慢病毒质粒载体序列和HPA基因序列设计用于无缝克隆的引物,HPA的PCR扩增产物与线性化的GV358质粒载体连接,获得重组慢病毒质粒。将经测序验证的重组慢病毒质粒与包装质粒共转染HEK-293T细胞,收取病毒感染液并感染HEL细胞,利用Western印迹法鉴定HPA过表达效果;利用流式细胞术检测细胞表面硫酸乙酰肝素(HS)的表达量;利用ELISA检测多配体蛋白聚糖1(syndecan-1)的释放量,以验证野生型和酶活缺失型HPA慢病毒表达载体的生物学效应。结果重组慢病毒质粒测序结果符合预期;Western印迹法检测结果表明,过表达HPA的HEL细胞有明显的HPA蛋白表达;流式细胞术结果显示,过表达野生型HPA的HEL细胞表面HS表达量的几何平均荧光强度(160.0±8.0)明显低于空载体对照细胞(345.0±15.2)(P<0.01);ELISA结果显示,过表达野生型HPA的HEL细胞培养基中多配体蛋白聚糖1的释放量(59.0±3.8)ng·L-1较空载体对照细胞(32.2±3.9)ng·L-1明显增多(P<0.01);而过表达酶活缺失型HPA的HEL细胞系细胞表面HS的表达量(353.0±14.0)和培养上清中多配体蛋白聚糖1的释放量(30.9±2.9)ng·L-1则与空载体对照细胞相当。结论成功构建了野生型和酶活缺失型的HPA慢病毒表达载体,并利用HEL细胞系对野生型和酶活缺失型HPA的生物学效应进行了验证,为研究HPA在机体病理生理过程中的作用机制提供了新的工具。

OBJECTIVE To construct lentiviral vectors overexpressing wild type and enzymatic activity deficiency heparanase(HPA)in order to find out more about the enzymatic effects and nonenzymatic effects of HPA.METHODS Primer sequences for seamless cloning were designed according to the vector sequence and gene sequence(wild type and enzymatic activity mutant:E225/343A).Then,linearized vector and genes amplified by PCR were recombinated.The highly purified recombi⁃nant plasmids were obtained by transformation,PCR identification,sequencing identification and plasmid extraction.The constructed expressing plasmids and packaging plasmids were co-transfected into HEK-293T cells,and cell culture supernates were collected and concentrated to obtain lentiviral infection solutions.HEL cells were infected with the virus and the expression of HPA was detected by Western blotting.The enzymatic activity of overexpressed HPA(wild type and enzymatic activity mutant)was validated by detecting the amounts of heparan sulfate(HS)on the cell surface using flow cytometry,while the amounts of syndecan-1 in cell culture supernates were detected using ELISA.RESULTS The sequencing results of recombinant plasmids were completely the same as expectated,and the overexpression of HPA was validated by Western blotting.As for enzymatic activity,flow cytometry results revealed that the Geometry mean fluorescence intensity of cell surface HS in HEL cells overex⁃pressing wild type HPA(160.0±8.0)was significantly lower than that of control cells(345.0±15.2),whereas its syndecan-1 levels in cell culture supernates(59.0±3.8)ng·L^-1 were higher than those of control cells(32.2±3.9)ng·L^-1.However,there was no significant difference in the amounts of HS(353.0±14.0 vs 345.0±15.2)or syndencan-1[(30.9±2.9)ng·L^-1 vs(32.2±3.9)ng·L^-1]between HEL cells overexpressing E225/343A mutated HPA and control cells.CONCLUSION Lentiviral vectors over⁃expressing wild type and enzymatic activity mutant HPA have been constructed and validated in HEL cell line,providing a novel HPA gene-editing tool for studying the physiological and pathological effects of HPA.