甜味蛋白Brazzein在Escherichia coli和Bacillus subtilis中的表达研究

通过实验对比研究了植物来源的甜味蛋白Brazzein单独及与E coli信号肽融合表达的情况,证实无信号肽序列的Brazzein编码基因实现了可溶性活性表达,它的肽链N端未融合其他氨基酸或肽链.同时,在Brazzein肽链中引入31His→Ala的突变,依照Bacillus subtilis的偏爱密码子,合成了定点突变后的Brazzein编码序列,并在B.subtilis WB600中实现了可溶性胞内表达,得到了表达量较高的活性蛋白产物.     

A novel gene <Emphasis Type=Italic>R049</Emphasis> identified in uropathogenic <Emphasis Type=Italic>Escherichia coli</Emphasis> provides partial protection in mice from colonization

Uropathogenic Escherichia coli (UPEC) is the most common causative organism of human urinary tract infection (UTI). Several UPEC virulence factors have been identified, but more are yet to be found. We previously identified a novel 789-bp-long DNA fragment (named R049) in UPEC strain 132 using a suppressive subtractive hybridization technique. In the present study, we used genome walking to elongate the sequence of this fragment to obtain the whole gene sequence and examined the role of this gene product in generating protective immunity. Through bioinformatic analysis, we predicted that this gene is a 1311-bp open reading frame (ORF), which we designated ORF_R049 (GenBank accession No.: EF488001). We further constructed a prokaryotic expression system to express full recombinant R049 protein and isolated and purified the protein through IPTG induction and nickel affinity chromatography. Using mouse immunosera generated by the purified protein, we confirmed the natural expression and outer membrane localization of the protein in wild-type strain UPEC132 by Western blotting. To test the potential of this protein as a vaccine candidate, we immunized mice with the recombinant protein before challenging them with UPEC132 through the urinary tract. The results showed significantly reduced bacterial colonization in the urine and kidneys of the immunization group compared with the control group. However, the degree of renal pathological damage was not significantly improved in the immunized mice. Our study has identified a novel gene of UPEC which can generate protective immunity against UTI. This novel gene provides a promising new vaccine candidate.     

响应曲面法优化重组人神经生长因子发酵培养基组成的研究

通过响应曲面法(RSM)达到优化重组人神经生长因子发酵培养基组分含量的目的.对发酵基础培养基采用四因素三水平实验设计,优化了葡萄糖、酵母粉、蛋白胨和KH2PO4的最佳浓度,得到了最大预测值并进行了验证.最终优化培养基组分为:葡萄糖7.95 g/L、蛋白胨19.06 g/L、酵母粉13.51 g/L和KH2PO43.87g/L,此时预测最大菌体量Ymax=2.398 g/L.验证试验结果表明:使用优化后的培养基表达重组人神经生长因子,其菌体量的平均值为(2.357±0.083)g/L,实验值与预测值基本相符,均高于优化前菌体量,且其表达量也得到相应提高.     

人纤溶酶原K1—3区基因在大肠杆菌中的表达

将人纤溶酶原Krigle 1-3(K1-3)基因插入融合表达载体pET-17b,获得重组质粒pET-K13,转化E.coli BI21(DE3),在IPTG诱导下,人纤溶酶原K1－3基因在E.coli BI21(DE3,pET-K13)中获得高效融合表达,表达量占菌体总蛋白的24％,表达产物以包函体存在,Western blot证明重组 蛋白对人纤溶酶原抗血清有特异免疫原性。     

论大肠杆菌的防治

大肠杆菌是动物肠道正常菌系的主要成员,为革兰氏阴性、中等大小的杆菌,无明显的夹膜,不形成芽孢,有鞭毛能运动,变异菌株无鞭毛不能运动。根据致病力特点分为:致病性菌株、条件致病性菌株和非条件致病性菌株。根据表面抗原O、K和鞭毛抗原H以及菌毛抗原F,大肠杆菌形成了复杂的血清型,在防治方面困难很大。大肠杆菌对热的抵抗力不强,对消毒药敏感,常规浓度短时间即可杀灭。     

纳米In2O3抗菌活性研究

采用最小抑菌浓度（MIC）和最小杀菌浓度（MBC）首次对纳米In2O3,的抗菌性能进行了研究．试验结果表明,纳米In2O3具有较好的抑菌和杀菌活性,对大肠杆菌的MIC为0．04ms／mL;对大肠杆菌的MBC为0．3mg／mL．时间动力学研究表明,纳米In2O3,与大肠杆菌作用30min后杀菌率达到99％以上．     

Evaluation of virus removal in MBR using coliphages T4

A membrane bioreactor (MBR) with gravity drain was tested for domestic wastewater for 65 days. Resultss howed that the effluent quality was excellent, and met with the reuse water standard of China (GB/T 18920-2002). Virus removal in the membrane separation process was investigated by employing coliphages T4 as a tracer. Two microfiltration membrane modules, with pore sizes of 0.22 and 0.1 μm, were used to investigate their effects on virus rejection at the transmembrane pressure of 8.5 kPa. It was found that 0.1 μm membrane had complete rejection of virus, and 0.22 μm membrane had significant rejection of virus. In the longterm operation of this MBR, no significant difference was observed between both pore sizes because the virus concentrations of the effluent in both cases were in the same order. Effluent virus concentration at steady state of MBR running was less than 2 PFU/mL. The removal ratios of coliphage T4 in MF processes were more than 10^5.5. The membrane surface deposits played an important role in the rejection of virus. The formation of cake clay on the membrane surface was the main cause of high rejection of colipbage T4 with MF of 0.22 μm.     

鸡大肠杆菌病致病因素及其研究进展

对鸡大肠杆菌致病力或毒力因子等相关致病因素作了简要概述,讨论了相关领域的研究进展.1）大肠杆菌的菌毛抗原是一种特异蛋白质,可以被免疫系统所识别,因此在疾病的免疫预防和相关疫苗研制等方面有着重要意义.2）鸡大肠杆菌肠毒素中LTB和STn亚单位具有良好的免疫原性,认为在降低ST的生物毒性有所突破的前提下,借用基因重组融合技术以及优化重组基因表达,是利用该毒素因子免疫防治的发展趋势.3）诸多实验研究表明,提纯的大肠杆菌OMP或通过基因工程表达的OMP同样具有良好的免疫原性和保护作用.某些细菌的不同血清型间有相同的OMP免疫原,这为OMP亚单位疫苗以至于基因疫苗的研发都提供了理论基础.     

大肠杆菌噬菌体的分离鉴定及生物学特性分析

利用野生型大肠杆菌MG1655为宿主菌,从下水道污水中分离得到一株噬菌体,编号为Phage 1.其噬菌斑大小为3～4mm,透射电镜观察发现该噬菌体有正多面体头部和弯曲尾部,属于长尾科噬菌体.对该噬菌体的生物学特性包括最佳感染复数、一步生长曲线、温度、氯仿、pH进行检测,结果显示该噬菌体的潜伏期为10min,繁殖周期为20min,对温度、氯仿以及pH耐受性良好.经基因组测序鉴定,该噬菌体为E.coli T1噬菌体.     

功能型DNA重组修复蛋白质RecR的体内分布示踪

RecR是参与大肠杆菌(E.coli)同源重组的蛋白质,在真核生物中存在功能保守性组分.通过构建recR_yfp 融合基因及其表达载体,对大肠杆菌菌体内RecR蛋白的分布情况进行了活体观察.显微镜观察结果表明,RecR蛋白一般在菌体的拟核区起作用.通过对比紫外线敏感性,Re-cR-YFP在重组质粒上提高了recR缺陷型大肠杆菌的抗紫外线能力,表明重组质粒表达的融合蛋白RecR-YFP具有生物活性,能够提高rPfR缺陷型大肠杆菌的存活率.