【作者】 杨晶；

【导师】 邓宁；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 目的:研究发现人类表皮生长因子域EGFL6可促进组织血管新生并在肿瘤组织细胞中表达上调，预示其与肿瘤发生发展密切相关，本研究通过克隆EGFL6全长及片段基因，构建pET32a(+)-EGFL6全长及片段蛋白原核表达载体，诱导表达并鉴定全长EGFL6蛋白及片段蛋白，并利用EGFL6片段蛋白免疫制备抗EGFL6多克隆抗体，应用其检测人肿瘤细胞系中EGFL6全长蛋白，为深入研究EGFL6的相关功能奠定了基础。方法:（1）全长人EGFL6基因的亚克隆与原核表达：以本实验室保存的EGFL6载体质粒为模板PCR扩增EGFL6全长基因，将其连接到pMDTM18-T经克隆，测序，酶切鉴定正确后连接至表达载体pET32a(+)上，转化到克隆菌株以及表达菌株，同样通过双酶切鉴定正确后,进行诱导表达，SDS-PAGE及Western blot的方法检测和鉴定目的蛋白的表达。（2）肿瘤细胞株中EGFL6的表达检测，从8株不同肿瘤及正常细胞系中提取总RNA，经反转录成cDNA后，利用实时荧光定量PCR方法检测8株细胞系中EGFL6基因的表达水平。（3）肿瘤细胞株中EGFL6基因片段的克隆与表达：以表达水平最高的一株细胞cDNA为模板进行PCR扩增EGFL6片段基因，将其连接到pMDTM18-T载体上经克隆,测序和酶切鉴定正确后连接至表达载体pET32a(+)上，转化克隆菌株Ecoli.DH5α及表达菌株E coliRosetta-gami B，同样通过双酶切鉴定正确后,进行诱导表达,SDS-PAGE及Western blot鉴定目的蛋白的表达。（4）EGFL6片段的纯化、多克隆抗体的制备与检测：目标蛋白经大量表达后，利用镍离子亲和树脂进行纯化EGFL6片段蛋白，以纯化产物为完全抗原免疫昆明小鼠制备抗EGFL6的多克隆抗体，该多克隆抗体经辛酸-硫酸铵法沉淀后纯化后，作为第一抗体对A375，SKOV3及HUVEC细胞裂解物进行Western blot检测分析。结果:（1）全长人EGFL6基因的亚克隆与原核表达：DNA序列测定及双酶切鉴定结果均显示EGFL6全长基因成功构建至原核表达载体pET32a(+)上，原核表达产物经SDS-PAGE及Western blot鉴定后证实EGFL6全长蛋白主要以包涵体的形式表达于大肠杆菌的周质腔内，并且包涵体表达量占EGFL6蛋白表达总量的90%以上。（2）肿瘤及正常细胞系中EGFL6表达水平检测：荧光定量PCR结果显示8株肿瘤及正常细胞系中，A375及SKOV3细胞的EGFL6表达水平明显高于其余细胞株。（3）肿瘤细胞株中EGFL6基因片段的克隆与表达：以A375细胞株总cDNA为模板，RT-PCR成功扩增EGFL6基因片段，DNA序列测定以及双酶切鉴定的分析结果均表明pET32a(+)-EGFL6片段基因表达载体构建成功。表达产物经SDS-PAGE及Western blot鉴定后证实EGFL6片段蛋白同时以包涵体和可溶性形式表达，并且各占表达总量的50%左右。（4）EGFL6片段的纯化、多克隆抗体的制备与检测应用：以纯化得到的EGFL6重组蛋白免疫制备得到小鼠血清，间接ELISA方法测得其效价为1：320000。Western blot结果表明,抗血清均能够特异地识别重组片段蛋白和重组全长蛋白。利用纯化后的抗EGFL6抗体对A375，SKOV3及HUVEC细胞裂解物进行Western blot检测，结果显示抗EGFL6多抗能特异性识别细胞系中天然EGFL6蛋白，同时， EGFL6蛋白在A375及SKOV3细胞系中表达量明显较HUVEC高，与前期荧光定量RT-PCR结果相符合。结论：从8株不同的肿瘤与正常细胞中筛选出高表达EGFL6的A375与SKOV3细胞，从A375细胞中克隆出EGFL6基因片段，并在原核表达系统中实现了EGFL6包涵体形式的全长蛋白表达及可溶性EGFL6片段蛋白的表达，并且进行了多克隆抗体制备及其初步检测应用。更多还原

【Abstract】 Objectives: The recent research showed that EGFL6was closely related to tumor genesisand development because of its angiogenesis promotion and over expression in most tumortissues and cells. In this study, the full-length EGFL6gene and EGFL6gene fragment werecloned and expressed in E.coli. The recombinant EGFL6fragment protein was purified andanti-EGFL6polyclonal antibody was prepared for detecting EGFL6in tumor cell lines, whichwill make foundation for EGFL6function study.Methods:(1).The sub cloning and prokaryotic expression of human full-length EGFL6:The full-length EGFL6gene was cloned by PCR amplification using pre-conserved EGFL6vector plasmid as a template, and then was constructed into pMDTM18-T followed byidentification with DNA sequencing and double enzyme digestion. The correct EGFL6gene wasthen constructed into expression vector pET32a (+) to express the full-length EGFL6proteinwith IPTG inducing. The EGFL6expression was analyzed by the SDS-PAGE and Western blot.(2)The detection of human EGFL6gene expression in cell lines: Real-time fluorescentquantitative PCR was used to detect EGFL6gene expression in6tumor cell strains and2normalcell strains.(3). The cloning of human EGFL6gene from tumor cells and expression in E.coli:The EGFL6gene fragment was cloned from the tumor cell with the high EGFL6expression, andconstructed in Prokaryotic expression vector pET32a (+) through gene recombination technology.The recombinant EGFL6was expressed with IPTG inducing, and the protein expression wereanalyzed by SDS-PAGE and Western blot.(4) The polyclonal antibody preparation and detectionof human EGFL6: The soluble EGFL6protein fragment was purified with Ni-NTA affinitychromatography. Kunming mices were vaccinated with the purified recombinant EGFL6and theanti-EGFL6polyclonal antibodies were prepared. The sensitivity and specificity of polyclonalantibody were analyzed by ELISA and Western blot. The antibody was purified through theCaprylic acid and ammonium sulfate precipitation for the further Western blot analysis in tumorcell lysates.Results:(1) The sub cloning and prokaryotic expression of human full-length EGFL6: Theresults of DNA sequencing and double enzyme digestion showed that the EGFL6full-lengthprotein expression vector pET32a (+)-EGFL6was constructed successfully. SDS-PAGE andWestern blot results showed that EGFL6whole protein was successfully expressed inRosetta-gami B stain of E. coli. The recombinant EGFL6was expressed mainly in inclusion body form, which was more than90%in the whole EGFL6protein.(2) The detection of humanEGFL6gene expressed in different cell strains: qRT-PCR detection results showed that EGFL6was highly expressed in tumor cell strains of A375and SKOV3.(3) The cloning of humanEGFL6gene from tumor cells and expression in E.coli: RT-PCR results showed that the EGFL6gene fragments were cloned successfully from A375cell strain, and the EGFL6expressionvector pET32a (+)-EGFL6was constructed successfully. SDS-PAGE and Western blot resultsshowed that the soluble EGFL6target protein was expressed at the size of38kD.(4) Thepolyclonal antibody preparation and detection of human EGFL6: Anti-EGFL6polyclonalantibody was prepared in Kunming mice and the Western blot analysis results showed that thepolyclonal antibody could recognize not only the recombinant full-length EGFL6or proteinfragment but also the nature EGFL6in cell strains. The western-blot results with polyclonalantibody showed that the EGFL6was highly expressed in A375and SKOV3and lowlyexpressed in HUVEC, which was corresponding to the qRT-PCR results.Conclusion: In this study, A375and SKOV3cell strains were selected to be the tumor cellstrains with high expression of EGFL6in8cell strains, and EGFL6gene fragment was clonedfrom A375cell. Full-length EGFL6and EGFL6protein fragment could be both expressed athigh level in E.coli. The specific polyclonal antibodies against EGFL6could be obtained bymice vaccination and used to identify and analyze EGFL6expression in cell lines, which can beused for further function studies of EGFL6.更多还原