猪繁殖与呼吸综合征病毒ORF5基因原核表达载体的构建与鉴定

本试验旨在构建能表达猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因的重组质粒。用疑似患猪繁殖与呼吸综合征(PRRS)病猪的脾脏、肺脏病料提取RNA,根据PRRSV ORF5基因设计1对引物,进行RT-PCR扩增,将ORF5目的基因克隆到pMD19-T载体中,得到pMD19-ORF5重组菌。根据测序结果和表达载体特点,设计1对带有NcoⅠ和XbaⅠ酶切位点的引物,扩增目的片段(dORF5),将其分别与pProEXHTb载体、pNZ8149载体连接,得到HTb-dORF5/DE3和pNZ8149-dORF5/NZ3900重组菌,分别用IPTG和Nisin诱导,再进行SDS-PAGE和Western blotting分析。结果显示,HTb-dORF5/DE3重组菌在1.5 mmol/L IPTG诱导时表达量最高,通过SDS-PAGE和Western blotting分析,在约22 ku处有特异性条带,且具有反应原性;pNZ8149-dORF5/NZ3900重组菌在20 ng/mL Nisin诱导时表达量最高,通过SDS-PAGE和Western blotting分析,在约19 ku处有特异性条带,间接免疫荧光试验有特异性绿色荧光。本研究成功构建重组质粒HTb-dORF5和pNZ8149-dORF5并获得表达,这为进一步研制预防PRRS疫苗奠定坚实基础。     

乳酸乳球菌的质粒消除

质粒消除是鉴定质粒和获得无质粒菌株的重要方法,是乳酸菌进行遗传学改造所需的一项重要技术。试验采用高温和消除剂结合的方法,对乳酸乳球菌镉抗性菌株进行质粒消除,探讨温度、消除剂吖啶橙的用量和作用时间对乳酸乳球菌镉抗性质粒消除的影响。结果表明,39℃高温可以质粒消除,而37和41℃均无此效果;独自吖叮橙作用未获得质粒消除菌株;39℃高温-吖啶橙同时作用比高温-吖啶橙交替作用消除率高,而39℃高温-20μg·mL-1吖啶橙共同作用12d,消除率可达98%。根据消除结果,以疑似功能性质粒为模板,进行PCR扩增,获得预期片段,进一步证实了其功能。     

表达猪传染性胃肠炎病毒S蛋白抗原表位乳酸菌对实验小鼠免疫功能的影响

为了探讨表达猪传染性胃肠炎病毒(TGEV)S蛋白抗原表位乳酸乳球菌对小鼠安全性及免疫功能,以口服的方式给实验小鼠免疫重组乳酸菌,观察小鼠的精神状态,采用ELISA试剂盒方法检测免疫指标。结果表明：重组乳酸菌对小鼠的精神状态及体重均无影响,能显著提高小鼠血清中IgG和粪便sIgA的含量。与对照组相比,免疫后1周、2周、5周的血清中IgG的含量差异显著(P<0.05),3周、4周差异极显著(P<0.01),表明能显著提高小鼠体液免疫水平;能显著提高外周血中T淋巴细胞增殖情况,与对照组相比,差异极显著(P<0.01),表明能显著提高小鼠细胞免疫水平。表达TGEV S蛋白抗原表位乳酸菌能显著提高小鼠体液免疫和细胞免疫水平,具有良好的免疫原性,且安全可靠。     

Drug resistance mechanism of the fish-pathogenic bacterium Lactococcus garvieae

The minimum inhibitory concentrations (MICs) of 15 chemotherapeutic agents were tested against 146 Lactococcus garvieae strains isolated from 1999 to 2006 in Japan. The agents used included chloramphenicol, ciprofloxacin, erythromycin (EM), enoxacin, fleroxacin, florfenicol, kanamycin, lincomycin (LCM), norfloxacin, oxolinic acid, orbifloxacin, ofloxacin, benzylpenicillin, streptomycin and tetracycline (TC). Of the tested strains, 46 showed high levels of resistance to EM, LCM and TC. Twelve of these strains were detected to be carrying transferable R-plasmids using a conjugation experiment and, using Southern hybridization, were shown to have the same structure as the R-plasmid. The remaining 34 resistant strains had a similar DNA structure to that of the R-plasmid as confirmed by polymerase chain reaction (PCR) using primers designed from sites in the transferable R-plasmid. The EM and TC resistance genes were classified into the ermB and tetS groups using PCR. We also detected gyrA and/or parC mutants that are highly resistant to old and new generation quinolones. This study revealed that transferable R-plasmids encoding EM, LCM and TC are widely distributed and are conserved regardless of the area and/or time of collection.     

幽门螺旋杆菌lpp20-cagA融合基因在乳酸球菌中的表达及免疫原性研究

为研究载体疫苗对幽门螺旋杆菌(HP)感染的免疫保护作用,本实验应用无缝克隆技术将HP的两个候选抗原基因lpp20和cagA直接连接后克隆于表达载体pNZ8149中,构建重组表达质粒pNZ8149-lpp20-cagA。将重组质粒电转化乳酸球菌(L.lactis)NZ3900,构建了不含任何抗生素筛选标记的食品级原核表达系统pNZ8149-lpp20-cagA/NZ3900,经nisin诱导表达,得到的重组蛋白分子量约为50.3ku。将以上得到的重组L.lactis灌胃免疫BALB/c小鼠,利用ELISA方法分别检测各组小鼠血清中IgG、IgG1、IgG2a及细胞因子IL-4、IFN-γ水平。结果显示,与对照组相比,实验组小鼠血清中IgG、IgG1、IL-4水平显著升高,表明重组蛋白Lpp20-CagA可以诱导良好的体液免疫应答;利用HP攻毒经重组菌灌胃免疫的小鼠,检测小鼠胃组织中HP的定植密度及脲酶活性,结果显示重组蛋白Lpp20-CagA在一定程度上可以减少HP在小鼠胃组织中的定植。本实验构建的载体疫苗在预防HP感染方面具有重要的应用价值。     

气相色谱法测定乳酸乳球菌脂肪酸

为了建立气相色谱法分析乳酸乳球菌脂肪酸方法,试验参照不同文献所提供的甲酯化方法对乳酸乳球菌进行甲酯化处理,通过对其气相色谱图中相同峰的峰面积进行比较,对比各个甲酯化方法的的效果。并对乳酸乳球菌MG1363进行气相色谱分析。结果表明,在对几种甲酯化方法进行比较后,发现酸碱结合法甲酯化法不适合乳酸菌,而三氟化硼甲酯化法明显优于酸催化与碱催化甲酯化法。并且测定出乳酸乳球菌MG1363的主要脂肪酸种类。结果表明,三氟化硼催化甲酯化法最适于乳酸乳球菌脂肪酸。乳酸乳球菌MG1363中除含有14和16C等脂肪酸外,还含有一种19C的环丙烷脂肪酸。     

Isolation and characterization of Streptococcus sp. from diseased flounder (Paralichthys olivaceus) in Jeju Island

Streptococcus sp. is gram-positive coccus that causes streptococcal infections in fish due to intensification of aquaculture and caused significant economic losses in fish farm industry. A streptococcal infection occurred from cultured diseased olive flounder (Paralichthys olivaceus) in May, 2005 at a fish farm in Jeju Island, Korea. The diseased flounder exhibited bilateral exophthalmic eyes and rotten gills; water temperature was 16~18℃ when samples were collected. Of the 22 fish samples collected, 3 samples were identified as Lactococcus garvieae and 18 samples were identified as Streptococcus parauberis by culture-based, biochemical test. Serological methods such as slide agglutination, hemolysis and antimicrobial susceptibility test were also used as well as multiplex PCR-based method to simultaneously detect and confirm the pathogens involved in the infection. S. parauberis and L. garvieae have a target region of 700 and 1100 bp., respectively. One fish sample was not identified because of the difference in the different biochemical and serological tests and was negative in PCR assay. In the present study, it showed that S. parauberis was the dominant species that caused streptococcosis in the cultured diseased flounder.     

乳酸链球菌最适抑菌培养条件的研究

为降低天然防腐剂乳酸链球菌素（Nisin）的生产成本,以金黄色葡萄球菌为试验菌,探讨了直接利用乳酸链球菌（Lactococcus lactis）培养液进行抑菌的最适培养条件。首先通过单因素试验,初步确定了最适碳源、氮源、金属离子及其浓度或配比的范围,然后采用正交试验L9（3^3）优化后得到的乳酸链球菌抑茵的最佳发酵条件为：3.0g／100g葡萄糖,4g／100g蛋白胨,2g／100g酵母膏,1g／100g牛肉膏,0.005mol／L MgSO4。优化后的培养基抑菌能力明显提高,此结果可为乳酸链球菌素的生产提供参考。