柯萨奇病毒B3 VP1蛋白的原核表达及免疫效果观察

发布时间：2017-12-27 11:10

本文关键词：柯萨奇病毒B3 VP1蛋白的原核表达及免疫效果观察　出处：《河北医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:柯萨奇病毒的B组(CVB)是人类病毒性心肌炎的主要病原体,以CVB3最为重要,迄今尚无有效的防治手段。VP1是CVB3的主要衣壳蛋白,含有多个T、B细胞表位,可诱导机体产生免疫应答。研究表明,编码VP1的DNA疫苗能诱导小鼠产生体液和细胞免疫应答,并对感染小鼠具有一定的保护能力,但不能有效阻止致死量病毒感染。推测与DNA疫苗在体内进入细胞的量较少,目的基因表达水平较低有关。研究表明,蛋白质有很强的免疫原性,免疫动物可诱导产生高效价的抗体。据此,许多人又重新尝试用蛋白质做为疫苗以提高免疫效果。此种疫苗是提取病原体中具有免疫保护作用的蛋白,或利用DNA重组技术使重组体表达这类蛋白、多肽或合成肽制成的疫苗,称为亚单位疫苗。亚单位疫苗不含有感染性组分,无须灭活,也无致病性。自l981年成功地将口蹄疫病毒的VP3基因克隆到大肠埃希氏菌内并制成用于牛和猪的疫苗之后,迄今已研制出包括乙型肝炎疫苗在内的许多亚单位疫苗用于传染病的预防。但也有研究表明,某些病原体亚单位疫苗的免疫原性低,不能达到满意的免疫保护作用。近年有研究显示,采用prime-boost方法将亚单位疫苗与DNA疫苗联合应用能明显提高疫苗的免疫效果。本研究用原核表达系统中表达CVB3 VP1蛋白,单独或采用prime-boost方法与CVB3 VP1 DNA疫苗联合应用免疫小鼠,观察免疫效果。方法: 1利用我室前期构建的真核表达质粒pcDNA3/VP1为模板,用VP1基因的特异性引物进行PCR扩增,将扩增产物与pGEM-T载体连接,转化入DH5α大肠杆菌,提取质粒进行酶切鉴定和测序筛选重组子。2经酶切鉴定和测序确认后,取双酶切的VP1片段和经过同样两种酶酶切的原核表达载体pET-his连接,构建原核表达质粒pET-his/VP1。3将原核表达质粒pET-his/VP1转入表达宿主菌BL21(DE3)pLysS,诱导表达携带有6×HIS标签的VP1蛋白,分别利用HIS单抗和CVB3多抗经Western-blot鉴定无误后对目的蛋白进行初步优化表达。将大量诱导表达后的菌体在冰浴中超声破碎,分离上清和包涵体沉淀,对该蛋白的水溶性进行分析。将包涵体溶液进行SDS-PAGE,切下目的条带,透析袋中透析。定量所得纯化后的蛋白。4用碱裂解法从转化的DH5α大肠杆菌中大量提取质粒pcDNA3/VP1,用聚乙二醇沉淀法进行纯化。5动物实验:将6～8周龄雄性BALB/c小鼠随机分为5组,分别为PBS对照组、pcDNA3/VP1质粒组、VP1蛋白组、pcDNA3/VP1质粒初免—VP1蛋白加强组(以下简称质粒蛋白组)和VP1蛋白初免—pcDNA3/VP1质粒加强组(以下简称蛋白质粒组),每组18只;纯化后的质粒用PBS稀释后,股四头肌注射免疫小鼠,纯化后的蛋白以PBS稀释后,腹腔注射免疫小鼠,初次免疫用完全弗氏佐剂,加强免疫用不完全弗氏佐剂。每次每只接种100μg;每3周免疫1次,共免疫3次;每次免疫后第14天,内眦静脉取血,分离血清,用微量中和试验(固定病毒-稀释血清法)检测血清中CVB3特异性中和抗体的水平;第3次免疫后3周,每组3只小鼠取脾脏制备淋巴细胞悬液,采用细胞计数试剂盒(cell counting kit-8, CCK-8)法进行特异性淋巴细胞增殖活性和特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)杀伤活性的检测;另每组12只小鼠用致死量的CVB3(4LD50)进行腹腔注射,观察并记录小鼠死亡情况至感染后第21天;每组剩余的3只小鼠用3LD50CVB3攻击,注射病毒后第7天处死,用于血中病毒滴度的测定。结果: 1用PCR从质粒中扩增出VP1基因片段,DNA测序证实此基因片段与报道的基因序列一致。2成功构建了原核表达质粒pET-his/VP1,双酶切结果证实插入和连接均正确。3在大肠杆菌BL21(DE3)pLysS中成功表达了CVB3 VP1蛋白,表达蛋白主要以包涵体形式存在。Western-blot证实表达的蛋白可与HIS单抗和CVB3多抗产生特异性反应。4不同免疫次数,各组血清中和抗体的平均效价分别为:PBS对照组未检测到;pcDNA3/VP1质粒组1:5.62,1:15.85,1:23.71;VP1蛋白组1:8.91,1:44.67,1:70.79;质粒蛋白组1:5.62,1:15.85,1:33.5;蛋白质粒组1:8.91,1:44.67,1:56.23。单因素方差分析表明,三次免疫后,除PBS组外,其余各组中和抗体滴度逐次增加(P0.05);三次免疫后,蛋白组、质粒蛋白组和蛋白质粒组中和抗体滴度高于pcDNA3/VP1组(P0.05)。5小鼠脾脏淋巴细胞增殖实验分别以CVB3和刀豆蛋A(concanavalin A, ConA)体外刺激淋巴细胞,检测细胞增殖活性。单因素方差分析表明,以CVB3刺激时,与PBS组和pcDNA3/VP1组相比,蛋白质粒组的增殖活性显著升高(P0.05);以ConA刺激时,蛋白组和蛋白质粒组均显著高于PBS组(P0.05)。6小鼠脾脏特异性CTL杀伤活性经单因素方差分析表明,质粒蛋白组在所有实验组中杀伤活性最高,PBS免疫组则最低,但两两比较无统计学意义。7用4LD50攻击小鼠,各组21天生存率分别为:蛋白组、质粒蛋白组和蛋白质粒组生存率达33.33%,而pcDNA3/VP1质粒组仅为8.33% , PBS组无存活。经χ2检验,蛋白组、质粒蛋白组和蛋白质粒组和对照组比较均差别有统计学意义。加强组小鼠的生存状况好于PBS组和pcDNA3/VP1质粒组(P0.05)。8与几个对照组相比,蛋白组、质粒蛋白组和蛋白质粒组小鼠血中病毒滴度显著降低(P0.05)。结论:1成功构建了CVB3VP1基因原核表达质粒pET-his/VP1。2成功表达、纯化出CVB3 VP1蛋白。3小鼠血清中和抗体滴度随免疫次数的增加而提高,三次免疫后,VP1蛋白参与的三种免疫方法诱导的和抗体水平高于DNA疫苗组。4蛋白-质粒免疫加强法能够增强小鼠淋巴细胞增殖活性和特异性CTL杀伤水平。5用致死量CVB3攻击小鼠后,蛋白组、质粒蛋白组和蛋白质粒组的小鼠生存率和存活时间均有所提高。
[Abstract]:Objective: the B group (CVB) of Coxsackie virus (Coxsackie virus) is the main pathogen of human viral myocarditis, and CVB3 is the most important. So far, there is no effective control method. VP1 is the main capsid protein of CVB3, which contains multiple T and B cell epitopes, which can induce immune response in the body. Studies have shown that the DNA vaccine encoding VP1 can induce humoral and cellular immune responses in mice, and has a certain protective ability to infected mice, but it can not effectively prevent fatal viral infection. It is presumed that the amount of DNA vaccine in the body is less, and the expression level of the target gene is relatively low. Studies have shown that protein has a strong immunogenicity, and immune animals can induce high - efficiency antibodies. On this basis, many people try to use protein as a vaccine to improve the immune effect. The vaccine is an immune protective protein extracted from pathogens, or a recombinant vaccine made of recombinant proteins, peptides or synthetic peptides by DNA recombination technology. It is called subunit vaccine. Subunit vaccines do not contain infectious components, without inactivation or pathogenicity. Since l981 successfully cloned the VP3 gene of foot-and-mouth disease virus into Escherichia coli and made it into the vaccine for Niu Hezhu, so far, many sub unit vaccines including hepatitis B vaccine have been developed for the prevention of infectious diseases. However, some studies have shown that the immunogenicity of some subunit vaccines of some pathogens is low and can not achieve satisfactory immune protection. In recent years, studies have shown that the combination of subunit vaccine and DNA vaccine by using prime-boost method can obviously improve the immune effect of the vaccine. In this study, CVB3 VP1 protein was expressed in prokaryotic expression system, and immunized mice were immunized with CVB3 VP1 DNA vaccine alone or by prime-boost, and the immune effect was observed.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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