截短型神经连接蛋白-1原核表达载体的构建、表达、纯化及其对阿尔茨海默症的初步治疗效果  

Construction of prokaryotic expression vector,expression,purification and preliminary curative effect on Alzheimer disease of truncated neuroligin⁃1

在线阅读下载全文

作  者:苏晓男 毕鹏翔 李超 黄青 于欣洋 吴丹[1,3] 郭艳芹 SU Xiao-nan;BI Peng-xiang;LI Chao;HUANG Qing;YU Xin-yang;WU Dan;GUO Yan-qin(Mudanjiang Medical University,Mudanjiang 157011,Heilongjiang Province,China;不详)

机构地区:[1]牡丹江医学院,黑龙江牡丹江157011 [2]牡丹江医学院附属红旗医院,黑龙江牡丹江157011 [3]牡丹江医学院医药研究中心,黑龙江牡丹江157011

出  处:《中国生物制品学杂志》2022年第11期1317-1325,共9页Chinese Journal of Biologicals

基  金:黑龙江省自然科学基金项目(H2017077);牡丹江医学院研究生创新科研项目(2019YJSCX-21MY);牡丹江医学院附属红旗医院“红旗科研基金”科研项目(2019HQ-08);黑龙江省中医药科研项目(ZHY16-019).

摘  要:目的克隆并构建5种截短型神经连接蛋白-1(tneuroligin-1,tNL-1)可溶性细胞外片段的原核表达载体,经表达、纯化后获得重组GST-tNL-1融合蛋白,初步探讨其对阿尔茨海默症(Alzheimer disease,AD)的治疗效果。方法对神经连接蛋白-1(neuroligin-1,NL-1)的结构特征及理化性质进行生物信息学分析。利用NL-1细胞外片段设计5个截短片段(tNL⁃1①~⑤),以NL-1细胞外片段1-691质粒为模板PCR扩增目的片段,构建pGEX-6P1-tNL⁃1原核表达载体,筛选最佳诱导条件后,表达重组tNL-1蛋白,经GST亲和层析法纯化重组蛋白。制备β淀粉样蛋白(β-amyloid protein,Aβ)寡聚体,并进行Western blot鉴定。将体外培养的人SHSY-5Y细胞随机分为3组:对照组(不加任何干预)、模型组(40μg/mL Aβ处理24 h)、实验组(Aβ与20、40、60μg/mL tNL-1②共处理24 h),MTT法检测tNL-1②对Aβ诱导的SHSY-5Y细胞的保护作用。结果NL-1有12个蛋白修饰位点,预测其中有8个修饰位点两两相关联,据此关联设计了5个截短体。质粒pGEX-6P1-tNL⁃1经双酶切及测序鉴定证明构建正确。最佳诱导条件为:37℃培养2 h,A600为0.8时降温至16℃,加入0.1 mmol/L IPTG诱导12 h。表达及纯化的重组tNL-1①~⑤蛋白相对分子质量分别约为68000、90000、66000、53000和62000,大小均与预期相符,且可被抗体特异性识别。制备的Aβ寡聚体在相对分子质量约4000和12000处条带明显,主要以单体和三倍体形式存在。MTT结果显示,与对照组相比,模型组细胞生存率明显下降(P=0.001);与模型组相比,实验组细胞生存率均明显升高(P<0.001)。结论Aβ对SHSY-5Y细胞存在毒性作用,制备的tNL-1能缓解Aβ对SHSY-5Y细胞模型的的增殖抑制作用。后续会将tNL-1蛋白应用于更多体内外试验中,为AD的治疗提供新思路。Objective To clone and construct prokaryotic expression vectors for soluble extracellular fragments of five kinds of truncated neuroligin-1(tNL-1),express and purify recombinant GST-tNL-1 fusion protein and preliminarily investigate its curative effect on Alzheimer disease(AD).Methods The structure characteristics and physicochemical properties of NLDOI:1 were analyzed by bioinformatics.The truncated segments tNL⁃1①~⑤were designed according to the NL-1 extracellular fragments,and the target fragment was amplified by PCR using the plasmid containing NL-1 extracellular fragment 1-691 as a template to construct prokaryotic expression vector pGEX-6P1-tNL⁃1.The condition for induction was optimized,based on which recombinant tNL-1 protein was expressed and purified by GST affinity chromatography.The oligomers ofβ-amyloid protein(Aβ)were prepared and identified by Western blot.Human SHSY-5Y cells cultured in vitro were randomly divided into control(untreated),model(treated with 40μg/mL Aβfor 24 h)and test(co-treated with Aβand 20,40 or 60μg/mL tNL-1②for 24 h)groups,and evaluated for the protective effect of tNL-1②on Aβ-induced SHSY-5Y cells by MTT assay.Results NL-1 had 12 protein modification sites,among which 8 were predicted to be pairwise associated,and therefore 5 truncates were designed based on the association.Plasmid pGEX-6P1-tNL⁃1 was constructed correctly as proved by restriction analysis and sequencing.The condition for induction was optimized as follows:culture the cells at 37℃for 2 h,drop the temperature to 16℃when A600 value was 0.8,and induce with 0.1 mmol/L IPTG for 12 h.The expressed recombinant tNL-1①~⑤after purification showed relative molecular masses of 68000,90000,66000,53000 and 62000 respectively,which were consistent with those expected and were recognized specifically by the antibody.The prepared Aβoligomers showed obvious bands with relative molecular masses of about 4000 and about 12000,and mainly existed in monomer and triploid forms.MTT assay showed that,compa

关 键 词:截短型神经连接蛋白-1 阿尔兹海默症 原核表达 纯化 突触 

分 类 号:Q81[生物学—生物工程]

 

参考文献:

正在载入数据...

 

二级参考文献:

正在载入数据...

 

耦合文献:

正在载入数据...

 

引证文献:

正在载入数据...

 

二级引证文献:

正在载入数据...

 

同被引文献:

正在载入数据...

 

相关期刊文献:

正在载入数据...

相关的主题
相关的作者对象
相关的机构对象