ASP-1重组蛋白的表达及其佐剂效应的研究

发布时间：2018-04-03 20:21

本文选题：ASP-1　切入点：佐剂　出处：《中国人民解放军军事医学科学院》2008年硕士论文

【摘要】： 本文研究的活化相关分泌蛋白-1(ASP-1, activation associated secreted protein-1)是一种来源于盘尾丝虫的蛋白,是一种非毒素类微生物衍生物型佐剂。目前已报道的初步实验研究显示,其具有良好的佐剂活性。本课题以ASP-1蛋白为研究对象,研究了其在原核系统表达的佐剂效应及特征,并对ASP-1蛋白在酵母系统中的表达及佐剂功能进行了初步的研究。一、ASP-1蛋白在原核系统中的表达及其佐剂活性分析 1、ASP-1基因的合成、克隆及序列分析根据Genebank登录的ASP-1序列,使用Upgene软件设计了36条引物(正反向各18条),采用重叠PCR方法,通过两轮PCR扩增,合成了ASP-1基因。产物克隆至T载体,双酶切后连接到pQE30载体中。经序列测定成功获得经过大肠杆菌密码子优化的目的基因。 2、ASP-1在原核表达系统中的表达、纯化和复性 ASP-1蛋白在原核大肠杆菌中以难溶的包涵体形式大量表达。经SDS-PAGE, Western-blot方法鉴定正确后,进一步将重组蛋白ASP-1通过镍离子柱进行亲和层析纯化,获得了高纯度的ASP-1蛋白。最终将纯化的ASP-1蛋白通过梯度透析的方法得到复性的可溶性重组蛋白。 3、以OVA模式抗原分析ASP-1的佐剂活性在对常用模式抗原卵清蛋白OVA免疫实验中,我们发现ASP-1佐剂组在诱导细胞和体液免疫应答水平均明显高于铝佐剂组和无佐剂对照组,尤其是相比较铝佐剂组在细胞免疫水平上对比更加明显。虽然OVA抗原最终仍是以Th2型免疫应答为主(IgG2aIgG1),但Th1型应答所占的比重明显升高,说明ASP-1表现出明显的同时诱导细胞与体液免疫应答的能力。 4、ASP-1蛋白在灭活疫苗联合免疫中的佐剂效应研究我们选择了三种常用灭活病毒疫苗:流行性出血热,流感和狂犬疫苗。结果表明,三联疫苗免疫时,不加ASP-1蛋白,IgG1/IgG2a滴度均不及或相当于各自单独免疫时水平;而加入ASP-1蛋白佐剂后,尽管各自用量减半,获得的免疫效果不论是Th1还是Th2应答方面甚至超过各自单独免疫的水平。说明了ASP-1佐剂的加入,可以达到减少疫苗各自的用量,诱导平衡Th1/Th2应答的目的。二、ASP-1在毕氏酵母系统中的表达及其佐剂活性分析在原核大肠杆菌中表达重组ASP-1蛋白的动物实验中,已经表明其具有良好的佐剂活性,但原核表达的包涵体难以溶解且复性过程烦琐,还可能丧失天然蛋白的某些活性。所以为了获得更加接近于天然结构的可溶性蛋白,我们采用了巴斯德毕氏酵母表达系统进行表达。首先利用Vector NTI软件设计出两条引物,将PCR扩增产物连接到T载体,通过双酶切的方法将目的片断ASP-1克隆到pPic9k载体中,PCR方法鉴定阳性克隆。随后将转化子转接到含有不同抗性浓度的G418培养基和MM、MD平板中筛选高拷贝株和不同的诱导表型。本实验酵母表达选择甲醇利用缓慢的Muts型来进行诱导表达。ASP-1蛋白在酵母中的表达分为两步,即菌体生长和蛋白诱导。先在以甘油为碳源的培养基上培养,当菌液OD值达到2-5后,将菌体悬浮于以甲醇为碳源的培养基中诱导表达。每隔24小时补加一次甲醇,分别在不同的时间段收集上清取样。样品最后经过SDS-PAGE, ELISA, Western-blot等方法进行鉴定。在用酵母表达的ASP-1蛋白进行功能性研究的过程中,我们同样选择了模式抗原OVA和狂犬灭活疫苗进行分析。初步结果显示,在与狂犬疫苗和OVA共同免疫BALB/c小鼠时,重组蛋白ASP-1表现出了较强的同时诱导细胞与体液免疫应答的佐剂活性。通过以上的研究说明,ASP-1蛋白具有良好的佐剂效应,有望发展成为一种新型的人用佐剂,值得深入研究。
[Abstract]:This paper studies the activation of secretory proteins associated with -1 (ASP-1, activation associated secreted protein-1) is a protein derived from Onchocerca volvulus, is a non toxin derivative microbial adjuvant. Preliminary experimental study has been reported that it has adjuvant activity. The ASP-1 protein as the research object. Study on the adjuvant effect and characteristics of prokaryotic expression system, and the expression of ASP-1 protein in yeast and adjuvant system were studied.
Expression of ASP-1 protein in the prokaryotic system and analysis of the activity of its adjuvant
1, ASP-1 gene synthesis, cloning and sequence analysis
According to the sequence of ASP-1 Genebank login, 36 primers were designed using Upgene software (positive and negative 18), using overlapping PCR method, through two rounds of PCR amplification, ASP-1 gene was synthesized. The products were cloned into T vector. After digested and ligated to pQE30 vector. After sequencing the success through the password. Optimization of Escherichia gene.
2, the expression, purification and renaturation of ASP-1 in the prokaryotic expression system
The expression of ASP-1 protein in prokaryotic Escherichia coli in inclusion insoluble form. By SDS-PAGE Western-blot method, after correct identification, the recombinant ASP-1 protein was purified by nickel ion affinity chromatography column, the purity of the ASP-1 protein. Finally the purified ASP-1 protein by gradient dialysis refolding obtained the soluble recombinant protein.
3, analysis of the adjuvant activity of ASP-1 by OVA model antigen
In the common mode OVA antigen ovalbumin immune experiment, we found that ASP-1 adjuvant group induced cellular and humoral immune responses were distinctly higher than that of aluminum adjuvant group and adjuvant control group, especially compared with aluminum adjuvant group in the cellular immunity level in contrast to the more obvious. Although OVA is still the final antigen with Th2 type immune response (mainly IgG2aIgG1), but the Th1 type response proportion increased significantly, indicating that ASP-1 also showed obvious ability to induce cellular and humoral immune responses.
Study on the adjuvant effect of 4, ASP-1 protein in inactivated vaccine combined immunization
We selected three kinds of inactivated virus vaccine, epidemic hemorrhagic fever, influenza and rabies vaccine. The results show that the triple vaccine, with ASP-1 protein, IgG1/IgG2a titers were less than or equal to separate immunization level; and ASP-1 protein added adjuvant, even though each half of the amount, whether it is the immune effect Th1 or Th2 response even more than their individual immune level. The addition of ASP-1 adjuvant, can reduce the dosage of vaccine induced Th1/Th2 response, balance.
Expression of two, ASP-1 in Pichia yeast system and analysis of the activity of its adjuvant
Animal experiment of recombinant ASP-1 protein expressed in prokaryotic Escherichia coli, had shown that it has good adjuvant activity, but the inclusion of prokaryotic expression and renaturation process cumbersome and difficult to dissolve, some activity may also lose the natural protein. So in order to get closer to the natural protein structure, we use the Pasteur Pichia expression system for expression.
We use Vector NTI software to design two primers, PCR amplification products connected to the T vector method by double enzyme digestion to fragment ASP-1 was cloned into pPic9k vector and the PCR method. The positive clones were identified and then transferred to the transformants with different resistance concentration of G418 medium and MM, MD in flat screen high copy lines and different induced phenotype.
The yeast expression of type Muts selection of methanol utilization slow to induce the expression of.ASP-1 protein expression in yeast is divided into two steps, namely, cell growth and protein induction. First cultured in medium with glycerol as the carbon source, when the OD value reaches 2-5, the cells were suspended in methanol to induce the expression of carbon source in the medium. Every 24 hours feeding time of methanol, the supernatant samples at different time respectively. Finally the sample after SDS-PAGE, ELISA, Western-blot and other methods were identified.
The process of functional studies in yeast ASP-1 protein expression, we also choose the mode of antigen OVA and inactivated rabies vaccine were analyzed. Preliminary results show that with rabies vaccine and OVA immunization of BALB/c mice, the recombinant ASP-1 protein showed strong activity with adjuvant induced cellular and humoral immune response.
According to the above studies, ASP-1 protein has a good adjuvant effect and is expected to develop into a new type of human adjuvant, which is worthy of further study.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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