鹅副黏病毒HN基因真核表达载体在小鼠中的免疫效果

以大量提取的鹅副黏病毒(GPMV)HN基因真核表达载体pVAXⅠ-HN和空载体pVAXⅠ肌肉注射BALB/c小鼠。利用MTT法检测免疫小鼠的T细胞增殖活性,用ELISA方法检测小鼠血清中GPMV抗体,观察GPMV HN基因真核表达载体pVAXⅠ-HN在小鼠中的免疫效果。结果表明:试验组小鼠血清中GPMV特异性抗体水平显著高于对照组,ELISA试验结果OD492值分别为1.360 0±0.101 7和0.358 0±0.018 1;试验组与对照组的淋巴细胞增殖试验刺激指数(SI)差异不显著,其SI值分别为1.181 2±0.030 1和1.119 1±0.035 5。由此说明,GPMV HN基因真核表达载体可以诱导小鼠产生明显的体液免疫。     

杂合抗菌肽Cec Md-CheRc的分子设计及其克隆载体的构建

为了获得具有高抗菌活性和低溶血活性的抗菌肽,利用家蝇抗菌肽Cec Md和中国林蛙抗菌肽Chensirin,设计出杂合抗菌肽Cec Md-Che Rc,并构其克隆载体。选取Cec Md的N端和Chensirin的C端氨基酸序列组成杂合肽,通过互联网和计算机软件,分析比较杂合肽的特征参数并进行结构预测。采用重复序列PCR方法合成2条杂合肽Cec Md-Che Rc I和Cec Md-Che Rc IV的基因,并连接到克隆载体pMD18-T上。分析发现杂合抗菌肽中Cec Md-Che Rc I疏水性最强,Cec Md-Che Rc IV净正电荷数最高;两者N端为疏水性α螺旋结构,C端为亲水性α螺旋结构;两者疏水性氨基酸大体分布在螺旋轮的一侧,其余氨基酸分布在另一侧;带正电荷的氨基酸主要分布在亲水面上。2条杂合肽的基因大小分别为133 bp和124 bp;PCR、双酶切和测序鉴定表明成功地构建了克隆载体pMD18-T/Cec Md-Che Rc I和pMD18-T/Cec Md-Che Rc IV,为后续研究奠定了基础。     

减毒鸡白痢沙门菌△aroA株的构建与致病性分析

以鸡白痢沙门氏菌c79-3强毒株为亲本,通过λ-red同源重组系统构建鸡白痢沙门菌aroA基因缺失株△aroA,并进行了测序验证和毒力试验。结果表明,aroA基因缺失株的生长速度较亲本株缓慢,且该缺失株具有良好的遗传稳定性;相比野生菌株,aroA基因缺失株的毒力发生了下降,雏鸡攻毒死亡率较亲本株低。本研究获得的减毒鸡白痢沙门氏菌aroA基因缺失株△aroA,为进一步研究鸡白痢沙门氏菌aroA基因的功能和后续减毒活疫苗开发奠定了基础。     

人HIF-1α基因重组减毒沙门氏菌载体的构建及稳定性检测

利用RT-PCR方法从低氧预处理的A549细胞中获得HIF-1α基因片段,将其定向插入pIRES-SEQ质粒NheⅠ、MLuⅠ2个酶切位点之间,得到真核表达载体pIRES-HIF-1α;采用电转化方法将其电转化入减毒沙门氏菌Ty21α中,获得重组减毒沙门氏菌菌株TPH;转染A549细胞,RT-PCR法检测HIF-1α基因表达,ELISA方法检测HIF-1α蛋白表达;TPH菌株在氨苄青霉素(+)和氨苄青霉素(-)LB培养板上分别传代40代,每10代提取质粒进行PCR及酶切鉴定.结果表明:试验成功构建了携带HIF-1α基因的重组减毒沙门氏菌TPH,其可在体外细胞中稳定表达HIF-1α基因及蛋白,TPH可稳定遗传.     

不同毒力传染性法氏囊病病毒衣壳蛋白酵母双杂交诱饵载体的构建及鉴定简

传染性法氏囊病病毒（infectious bursal disease virus,IBDV）毒株Gx（超强毒株）、F9（中等毒力毒株）、Gt（弱毒株）遗传背景高度相似,但生物学性状差异显著。为研究不同毒力毒株与宿主细胞相互作用的分子细节,本研究将IBDV Gx、F9、Gt毒株的衣壳蛋白VP2基因克隆入pGBKT7载体,分别构建了诱饵载体pGBGxVP2、pGBF9VP2和pGBGtVP2。经Matchmaker Gold Yeast Two-hybrid System验证,结果显示所构建的3个诱饵载体均无自激活作用,对酵母细胞无毒性作用。本研究为利用酵母双杂交系统深入研究IBDV与宿主相互作用奠定了基础。     

Identification of eggs from different production systems based on hyperspectra and CS-SVM

1. To identify the origin of table eggs more accurately, a method based on hyperspectral imaging technology was studied.

2. The hyperspectral data of 200 samples of intensive and extensive eggs were collected. Standard normalised variables combined with a Savitzky–Golay were used to eliminate noise, then stepwise regression (SWR) was used for feature selection. Grid search algorithm (GS), genetic search algorithm (GA), particle swarm optimisation algorithm (PSO) and cuckoo search algorithm (CS) were applied by support vector machine (SVM) methods to establish an SVM identification model with the optimal parameters. The full spectrum data and the data after feature selection were the input of the model, while egg category was the output.

3. The SWR–CS–SVM model performed better than the other models, including SWR–GS–SVM, SWR–GA–SVM, SWR–PSO–SVM and others based on full spectral data. The training and test classification accuracy of the SWR–CS–SVM model were respectively 99.3% and 96%.

4. SWR–CS–SVM proved effective for identifying egg varieties and could also be useful for the non-destructive identification of other types of egg.     

人血小板因子Ⅳ在家蚕杆状病毒表达载体系统中的表达

将人血小板因子Ⅳ (HumanPlateletFactorⅣ ,简称hPF4)基因重组于家蚕杆状病毒转移载体pBacPAK8中 ,获得重组转移载体pBacPAK PF4,并与线性化病毒Bm BacPAK6DNA共转染家蚕培养细胞 ,在细胞内发生同源重组 ,获得重组病毒BacPAK PF4。Southern杂交结果表明重组病毒基因组中含有hPF4基因。重组病毒以MOI=10感染家蚕培养细胞 (2× 10 6个细胞 )和家蚕 5龄幼虫 ,表达产物用体外培养的血管内皮细胞测定其生物活性 ,测得表达量在家蚕培养细胞中第 3天达到最高值为 6 0 88μg/ 2× 10 6个细胞 ;在蚕体内表达第 5天达到最高值 ,表达量明显高于家蚕培养细胞     

猪白细胞介素2基因的克隆及腺病毒表达载体的构建

根据GenBank中收录的猪白细胞介素2(pIL-2)基因序列,设计1对特异性引物.运用RT-PCR技术从刀豆素(Con A)诱导的猪外周血淋巴细胞总RNA中扩增得到pIL-2基因,并将其克隆至pGEM-T Easy栽体上.核苷酸测序结果显示,获得的基因序列全长493 bp,含465 bp的开放阅读框,编码154个氨基酸,与已知pIL-2核苷酸序列和氨基酸序列的同源性都高达100%.将克隆的pIL-2基因编码阅读框亚克隆至腺病毒穿梭栽体pShuttle-CMV,电转化含腺病毒基因组(pAdEasy-1)的大肠埃希茵细胞BJ5183-AD-1,成功获得了重组腺病毒DNA,将纯化后的重组腺病毒DNA转染AD-293细胞,经过病毒基因组的PCR鉴定和转录水平的RT-PCR鉴定,初步表明,获得了表达pIL-2的重组腺病毒.     

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.     

基于主成分分析和遗传支持向量机的发动机故障诊断

为了实现发动机故障的快速实时诊断,提出一种基于主成分分析（PCA）和遗传支持向量机（GA-SVM）的发动机故障诊断方法。该方法利用振动信号经小波变换和主元分析来提取故障特征,以减少信号的冗余。针对人为选择SVM参数的盲目性,应用遗传算法优化其参数,并与BP神经网络（BPNN）比较。试验结果表明：GA-SVM比BPNN具有更强的分类识别能力,小样本故障诊断正确率达100%。