【作者】 舒红；

【导师】 任常山；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2003， 博士

【摘要】 目的 神经生长因子（NGF）作为神经营养因子家族的一员，对神经元存活、生长、发育与分化、损伤修复与再生等方面具有重要作用，它还参与免疫反应，具有促进创伤愈合和血细胞生长，促进肿瘤分化、抑制肿瘤生长等作用。由于天然的NGF来源少，不能满足临床和科学研究的需要，因此，用基因重组技术来获得大量高活性的人NGF蛋白已成为当务之急。本研究对人含前导肽的β-NGF基因序列进行PCR扩增，直接插入pGEM-T载体进行亚克隆，并对所构成的重组子pGEM-Tβ-NGF进行分析鉴定。把亚克隆的β-NGF基因插入分泌型酵母表达载体pPIC9K中，构成重组表达质粒pPIC9Kβ-NGF，将其转化入Pichia pastoris酵母GS115菌株中进行表达，以获得具有高生物活性的神经生长因子蛋白，为下一步临床应用打下基础。 方法 （1）用酚/氯仿/异丙醇方法从人胎盘中提取总DNA。 （2）采用PCR技术，根据β-NGF基因序列设计一对特异引物，用Taq+pfu DNA聚合酶从人胎盘DNA基因组中扩增出含前导肽及信号肽的β-NGF基因序列。 （3）采用A-T克隆法，在T₄DNA连接酶存在的条件下，把从PCR产物中纯化所得到的β-NGF基因与pGEM-T载体直接连接后，转化入用氯化钙法制备的JM109感受态菌中，经蓝白斑试验筛选，得到白色的阳性转化菌株。转化菌株经过扩大培养后，经提取后得到重组质粒pGEM-Tβ-NGF。用双酶切法、PCR法及序列分析对重组子pGEM-Tβ-NGF进行鉴定。 （4）将亚克隆所得到的重组质粒pGEM-Tβ-NGF和酵母表达载体pPIC9K分别同时用内切酶SnaBⅠ和AvrⅡ进行双酶切，所得到的两段目的片段进行连接，得到重组表达质粒pPIC9Kβ-NGF。用SacⅠ对其进行线性化，用脂质体法将其导入GS115中，在组氨酸缺失的RD平板上筛选出阳性菌株。用PCR法对这些菌株进行筛选和Mut+/Mut‘表型的鉴定。 （5）选取含有Mut十的转化子的单菌落，放人以甲醇为唯一碳源的BM-MY培养液中，培养3一4天，每24小时加人一次甲醇，使终浓度为0.5%，以维持甲醇的浓度，诱导其表达和分泌蛋白。 （6）用SDS一PAGE电泳对表达蛋白进行分析，用Westem blotting方法对所表达蛋白进行免疫活性鉴定。结果 （l）从人胎盘中提取的总DNA，其OD，/OD280“1 .8。 （2）用一对特异引物，采用PCR法，从人胎盘总DNA中扩增出的产物，经1.2%的琼脂糖凝胶电泳，结果显示，在748bp处有一条阳性目的条带。对其纯化产物进行双向测序，结果显示与文献报道的p一NGF序列相同，未发生碱基的突变。 （3）亚克隆中，从蓝白斑试验中所得到白色转化菌株中提取的重组质粒pGEM一T日一NGF，经h吐I酶切（pGEM一T载体上有2个乃瓜11酶切位点）后，得到了预期的2564bp和1 185bp两条带;用这些白色菌落分别作为PcR反应的模板，反应产物经1 .2%的琼脂糖凝胶电泳显示在748位置上出现了阳性条带;用，I7及SP6作为测序引物，对重组子pGEM一Tp一NGF进行测序，其碱基序列与文献报道相同。 （4）在叭DNA连接酶作用下，带有互补粘性末端的p一NGF和pPIcgK连接后，形成重组质粒nPICgKp一NGF。用。一factor和3’Aoxl引物（儿t中提供）作为测序引物，对nPICgKp一NGF进行双向测序。结果表明，p-NGF基因已正确插人pPICgK载体，序列与文献相同，没有突变和错配，且其翻译启动子已正确插人pPICgK载体的阅读框架。 （5）为了增加外源DNA的同源整合机率，用Sacl线性化重组表达质粒pPICgKp一NGF。，用脂质体法将其导人酵母菌株Gsl巧。阳性菌株筛选和Mut+/Mut‘表型的鉴定采用PCR法。以5‘AOXI及3‘AOXI序列引物为引物的PCR产物，如果在出现1 .ZKb左右的包含插人子在内的阳性片段的同时，在2.ZKb的位子上也出现一条带，说明宿主菌的AOXI基因未被破坏，为Mut+表型，反之，为Mut.表型。 （6）4个Mut+性菌落在BMMY培养液中经30℃振摇、甲醇诱导后，所表达的蛋白经SDS一PAGE电泳染色显示，72h及%h的上清样品在14.OKD左右有一条特异条带。 （7）用Westem bfotting鉴定，采用兔抗鼠NGF多克隆抗体进行免疫组化染色，显示在14.OKD有特征带。经积分扫描分析，表达目的蛋白占细胞外泌总蛋白的30%。结论 （l）用酚/氯仿/异丙醇方法提取的人胎盘DNA纯度较高。 （2）用Taq+nfu DNA聚合酶所扩增出来的PCR产物可靠，未出现碱基突变。 （3）用CaCI:法制备的JM109感受态菌有很好的转化率。 （4）亚克隆的重组质粒pGEM一Tp一NGF构建成功。 （5）制备的脂质体具有较好的转化能力。 （6）重组表达质粒pPICgKp一NGF构建成功。 （7）用PCR法同时对阳性转化菌株的筛选和Mut+/Mut，表型的鉴定具有简便、可靠的优点。 （8）piehia pastoris酵母GSI 15菌株对p一NGF蛋白的表达在72小时以后开始稳定表达。 （9）Pichia Pastoris酵母所表达的自身的外泌蛋白较少，使分泌的目的蛋白易于提纯。 （10）Pichia pastoris酵母所表达的蛋白有较高的表达率。 (川所表达的p一NGF蛋白具有免疫活性。更多还原

【Abstract】 ObjectiveNGF was one of the nerval trophic factors, it played an important role in the survival, growth, development, differentiation, repair and regeneration of nerve cells. Meanwhile, NGF was also thought to have a profound effect on im-munoreaction.NGF has being had more and more effects on many kinds of diseases, such as Alzheimer’s and Parkinsonian’s disease. The recent studies demonstrated that NGF also had some effects on such diseases as peripheral nerve impairment, optic nerve disease, brain trauma and so on.The amount of NGF was less in nature. So, it was imperative to obtain an amount of active NGF by the gene - recombined technology.The present study was designed to obtain the pro - - NGF gene by PCR amplification, the recombinant pGEM -T -NGF by subcloning, the active - NGF protein by inserting the gene into the expression vector pPIC9K and transforming into the Pichia pastoris stain cells GS115.Methods(1) The pro - (3 - NGF gene was amplified by using PCR with a pair of specific primers designed in according to the - NGF gene sequence and Taq plus pfu DNA polymerase.(2) The recombined plasmid pGEM - T - NGF was obtained by A - T cloning method. PCR product was ligated directly with pGEM - T vector to be formed the recombinant pGEM - T - NGF and the mix was transformed into the competent E. coli cells (JM109) that was done by Calcium Chloride method.We plated the transforming mix onto LB plates with Ampicillin/ X - gal/IPTG. The transformations were analyzed by Blue - White Spot Assay. The white spots were detected by enzymolysis, PCR and sequencing respectively.(3) The recombinant pGEM - TfJ - NGF and expression vector pPIC9K were digested doubly with SnaB I and Avr H respectively. The aimed DNA was isolated by 1.2% argarose gel electrophoresis, and purificated by Gel DNA Abstraction Kit. The p - NGF was ligated with pPIC9K by T4DNA ligase and was transformed into competent E. coli cells JM109.(4) The positive colonies from the transformed cells growing on the LB/ Amp plate were picked into LB medium with Amp, and the recombinant pPIC9K- - NGF was abstracted using Plasmid DNA Mimiprep Kit, and its sequencing was performed with primers -factor and 3’AOX1.(5) Both pPIC9K - NGF and the parent vector pPIC9K was linearized by Sacl. Then, they were transformed into Pichia pastoris strain, GS115, by spher-oplasting. The spheroplast was spread onto the His- RDB plates. The positive colonies were obtained from the His ~ RDB plates and spread onto YPD plates. To identify the Mut+ /Mut" phenotype, positive colonies were pickde and acted as templates. The PCR was performed with the primers 3’AOXl and 5’AOXl.(6) The recombinant strain was inoculated with RDB medium at 30^Cwith shaking at 200rpm and induced by 100% methanol ( final concentration 0.5% ). The methanol was added every 24 hours. Supernatant at 24, 48, 72, 96 hours was obtained in order to be analyzed.(7) The expression supernatant was analyzed using SDS -PAGE electrophoresis and Western blotting with rabbit - anti - rat polyclone NGF antibody.Results(1) The 1. 2% agarose gel electrophoresis results of PCR showed the 748 bp gene fragment was obtained from abstraction of placenta DNA and amplification with a pair of special primers. The sequence of (3 - NGF gene obtained was the same as the reported.(2) In the Blue - White Spot Assay, the white colonies were positive ones that contained the recombinant. After the recombinant pGEM - Tp - NGF was purified and digested by Pvu II , the results of 1.2% agarose gel electrophoresis showed that the positive bands appeared in both 2564bp and 1185bp.(3) White colonies were acted as templates for PCR. Their PCR products were analyzed by 1.2% agarose gel electrophoresis, which revealed that the positive band appeared in 748bp. The recombinant pGEMT - P - NGF sequence was the same as the reported.(4) Both pGEM - Tp - NGF and pPIC9K contained SnaB I and AvrH restriction site. The recombinant pPIC9Kp - NGF was obtained throu更多还原