【作者】 吕宁；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 发育生物学， 2007， 硕士

【摘要】 猪传染性胃肠炎(Transmissible gastroenteritis of swine,TGE)是由猪传染性胃肠炎病毒(Transmissible gastroenteritis virus of swine, TGEV)引起的一种高接触性传染病。在TGEV编码的四种结构蛋白中,只有纤突蛋白(S蛋白)能够诱导中和抗体的产生。而位于S蛋白近N端A、B、C、D四个主要抗原位点片段诱导产生的免疫保护作用,几乎相当于整个S蛋白产生的诱导效应。因此,对于S蛋白抗原位点区的研究可以为TGE的诊断,预防提供一定的基础条件。本研究以S基因抗原位点区作为目的基因,将其克隆至原核表达载体pET-28a中,并转化入大肠杆菌BL21(DE3)菌株后,以IPTG诱导目的蛋白的表达。表达产物经SDS-PAGE电泳及Western-blot检测均与预期结果相符的基础上,利用自动诱导表达系统进一步提高目的蛋白的表达量。相关结果如下:1.以带有全长S基因的质粒pGS为模板,通过PCR扩增2.1 kb的S基因抗原位点片段S1。并将其定向克隆至原核表达载体pET-28a中,构建了pET-S1原核表达重组质粒。2.将带有重组质粒pET-S1的BL21(DE3)菌株,分别于37℃、30℃、28℃下诱导培养5 h后,收集菌液稀释105倍,涂布含Kanar和无抗性的LB平板,经菌落计数表明,在37℃和30℃诱导时,分别约有60 %和30 %的大肠杆菌细胞丢失重组质粒;而28℃诱导则pET-S1在大肠杆菌BL21(DE3)中可较稳定存在。SDS-PAGE电泳显示,37℃、30℃诱导条件下蛋白表达不明显,而将诱导温度降低至28℃时可明显提高目的蛋白的表达。3.在28℃条件下以IPTG诱导目的蛋白表达。经SDS-PAGE电泳及Western-blot检测表明,目的蛋白产物大小与预期一致为76 ku,并且具有天然蛋白抗原性。通过凝胶分析软件Bandscan分析显示,随培养时间的增加,目的蛋白表达量逐渐增加,最高约占细菌总蛋白的25.9 %。未诱导的对照组也有少量目的蛋白的表达,约占菌体总蛋白的3.3 %。4.分别设置pET-S1转化菌的诱导组和未诱导组,通过测定菌液振摇培养相同时间的OD600值表明,未诱导组在振摇培养初期菌液的OD600值可达到3.65,最高可达到6.19;而诱导组菌液的OD600值最高只可达到2.68,由此说明诱导开始后菌株的生长在一定程度上受到了限制。5.利用自动诱导表达系统,通过重新定义菌株复苏生长以及目的蛋白诱导表达时的培养基组分,使目的蛋白表达量提高到45.1 %。6.以IPTG诱导作为对照,pET-S1转化菌在自动诱导培养基中振摇培养12 h。菌液的OD600值和pH值表明,在IPTG诱导体系中,菌液OD600最高仅达到2.63,并且随诱导时间增加,菌液有逐渐偏碱性的趋势;反之在自动诱导体系中,菌液的OD600值最高可达到12.65。而pH值在振摇培养过程中基本接近中性。更多还原

【Abstract】 Transmissible gastroenteritis of swine (TGE) which is caused by transmissible gastroenteritis virus of swine (TGEV) is an acute, highly prevalent enteric infectious disease. TGEV have four major structural proteins. The major antigenic sites of TGEV involved in the induction of virus neutralizing antibodies are located in the globular portion of the spike (S) protein. The S protein of TGEV has four major antigenic sites, which can induce immune protecting effects as same as the whole S protein does. So it is vital to get a DNA fragment including S gene whole antigenic sites in swine transmissible gastroenteritis coronavirus in order to prevent and treat TGE.In this research, the DNA fragment including S gene whole antigenic sites was cloned and inserted into pET-28a vector. The recombinant plasmid transformed into E.coli strain BL21 (DE3) to induce the expression of S protein with IPTG. SDS-PAGE and Western-blot indicated that the expression of target protein was the same as the expected results. Through auto-induction expression system, the amount of target protein was improved significantly. The results of research are as following:1. A DNA fragment including S gene whole antigenic sites in swine transmissible gastroenteritis coronavirus which was 2.1 kb was amplified by PCR. The recombinant expression plasmid pET-S1 was constructed by inserting target gene into pET-28a vector.2. The recombinant plasmid pET-S1 was transformed into E.coli strain BL21 (DE3) to induce the expression of target protein under 37℃, 30℃and 28℃. Stability of cultures was tested by plating on agar plates after 105-fold dilution. The results indicated that 60 % and 30 % E.coli strain lost pET-S1 when strain was induced under 37℃and 30℃, respectively. The recombinant plasmid can maintain stability under 28℃.3. The recombinant plasmid pET-S1 was transformed into E.coli strain BL21 (DE3) to induce the expression of target protein under 28℃. The result of SDS-PAGE and Western-blot indicated that the target protein was about 76 ku which is the same as the expected length and maintained antigenicity of TGEV. The highest amount of target protein accounted for 25.9 % of total bacteria protein. 3.30 % target protein can be detected in non-induced E.coli cultures.4. The results of optical density at 600 nm (OD600) of non-induced cultures and induced cultures demonstrated that the saturating density of non-induced cultures doubled the induced cultures to OD600~6.19.5. Through auto-inducting expression system, the recombinant plasmid transformed into E.coli strain BL21 (DE3) which was cultured in the changed culture medium and condition. the highest amount of target protein accounted for 45.1 % of the total bacteria protein.6. In IPTG-induction systerm, the highest OD600 of cultures was 2.63, and cultures had a trend of turning to alkaline. While in auto-inducing systerm, the highest OD600 of cultures was 12.65, the cultures can maintain neutral.更多还原