传染性法氏囊病病毒广西分离株血清亚型的确定

应用兔制备的抗传染性法氏囊病病毒(IBDV)高免血清,在鸡胚成纤维细胞(CEF)上对从广西发病鸡群分离的4个IBDV代表性流行毒株和2个常用疫苗株进行交叉中和试验。结果表明6个毒株被分为2个血清亚型;根据试验所得的R值,应用聚类分析法分析了各亚型毒株之间的亲缘关系,结果显示目前在广西流行的IBDV野毒株之间以及其与疫苗株间的抗原性存在一定的差异。研究结果对及时掌握广西IBDV流行毒株的抗原变异并为研制更有效的适合本地使用的IBD疫苗提供了科学依据。     

口蹄疫病毒VP1基因的原核可溶性表达

针对重组原核表达载体表达目的蛋白以包涵体形式存在的问题,作者对影响外源蛋白表达的IPTG的浓度、温度、时间等因素均进行了探索,以观察VP1蛋白的可溶性情况。通过PCR技术将VP1基因克隆至pGEM-T载体中,然后将pET-28a(+)和pGEM-T载体分别进行双酶切,构建重组的pET-28a-VP1载体,将重组载体转化至BL21中,诱导后经SDS-PAGE检测可见约29 ku的目的蛋白条带。插入的外源目的蛋白主要以包涵体的形式存在,上清中的可溶性蛋白甚微。试验结果表明,这可能与蛋白本身的氨基酸组成有重要关联。     

鸡Mx基因的克隆与原核表达

本研究旨在通过克隆鸡Mx全基因序列进而进行该基因的原核表达,获得具有生物学活性的蛋白.利用poly Ⅰ:C诱导鸡胚成纤维细胞Mx基因表达,克隆了Mx基因全长cDNA序列,将开放阅读框(ORF)连接构建于表达质粒pGEX-4t-2中获得重组表达载体pGEX-Mx,转化Rosetta(DE3)菌株,经IPTG诱导后检测.表达产物检测显示该蛋白的相对分子量为75 ku.说明获得了Mx基因的高效表达,为进一步进行Mx基因的活性检测以及利用Mx蛋白进行抗病毒转基因的研究奠定了基础.     

用响应面分析法优化桑叶水溶性多糖的提取工艺

为了高效提取桑叶中的水溶性多糖,通过单因素试验考察了提取温度、提取时间、原料质量浓度和提取次数对多糖得率的影响,然后采用响应面分析法优化桑叶水溶性多糖的提取工艺。以提取温度和原料质量浓度为主要因素进行中心组合试验,建立了多糖得率测算模型的回归方程,确认提取温度和原料质量浓度对多糖得率都有显著影响。优化后的提取工艺在温度90℃、提取时间80 min、原料质量浓度0.042 g/mL的条件下提取1次,桑叶水溶性多糖的最高得率达到1.92%。     

甘肃沙枣多糖提取工艺研究

采用热水浸提、醇沉法提取沙枣多糖,对沙枣多糖提取工艺进行了研究。选择料液比、浸提温度和浸提时间作为单因素进行梯度实验,确定其提取备件范围,再通过正交试验进一步确定沙枣多糖最佳提取工艺条件。结果表明,沙枣多糖的最佳提取工艺为：浸提温度为85℃、固液比为1：60、浸提时间为2h,在此条件下提取得甘肃沙枣多糖含量为4．98％。     

蓝黄青口服液对鸡传染性喉气管炎疫苗免疫的影响

为测定蓝黄青口服液(LHQY)对鸡传染性喉气管炎(ILT)免疫以及红细胞免疫力的影响,试验分为4组,分别为对照组、LHQY(浓度为1∶1500)饮水组、鸡传染性喉气管炎和鸡痘基因工程活载体疫苗免疫组和二者同时给予组.结果显示,LHQY给药后3d就能极显著提高健康鸡的RBC-CR1花环率和RBC-IC花环率(P＜0.01),并且这种作用效果可以持续20d以上;与疫苗同用,RBC-CR1花环率和RBC-IC花环率均显著或极显著高于健康对照组和疫苗组(P＜0.05,P＜0.01).进行攻毒保护试验,发现LHQY用药组的RBC-CR1花环率和RBC-IC花环率均极显著高于感染对照组(P＜0.01),LHQY与疫苗混合使用这两种花环率也极显著高于感染对照组(P＜0.01),并整体高于疫苗组.说明蓝黄青口服液能显著提高鸡红细胞免疫功能,对ILT有很好的预防和治疗作用.     

IPTG诱导浓度、时间对AsiaⅠ型口蹄疫病毒VP1蛋白表达的影响

将口蹄疫病毒VP1基因插入原核表达载体pET-28a中,转化至大肠杆菌BL21.用不同的IPTG浓度与不同诱导时间诱导菌株表达VP1蛋向,分析不同IPTG浓度及不同诱导时间对蛋白表达量的影响,以确定最佳表达条件.结果表明,在一定范围内,随IPTG浓度的增高,表达量并不会随之增大;而在菌体对数生长期内,随诱导时间的延长,表达量会随之增大.最后确定当IPTG浓度为3 mmol/L,诱导表达到最长时间9 h时,VP1蛋白表达量最大.     

30株鸡大肠杆菌ESBLs基因型检测及耐药性分析

采用2倍稀释法检测30株鸡大肠杆菌对常见药物耐药性,用表型筛选和确证试验检测产超广谱β-内酰胺酶(ESBLs)情况,并分别用TEM、SHV和CTX-M等3种通用引物进行PCR扩增以确定其基因型.结果显示23株大肠杆菌为产ESBLs株,占76.7%.PCR扩增表明TEM、CTX-M、SHV阳性率分别为73.9%、43.5%和0%,有7株同时检测出TEM和CTX-M基因(占30.4%),3株未检测出TEM、CTX-M和SHV 3种基因(占13.0%).药敏试验结果显示产ESBLs大肠杆菌对药物的多重耐药性明显比非产酶大肠杆菌严重,产酶菌对氨苄西林和头孢噻呋的耐药率最高,为95.7%,对氨基糖苷类、恩诺沙星和复方新诺明的耐药率均高于78%,氨苄西林和三代头孢与酶抑制剂联用或2种不同种类的药物联用均能明显增强抗菌活性.以上结果表明河南省76.7%的临床分离鸡大肠杆菌为产ESBLs株,其基因型主要是TEM和CTX-M,尚无SHV型,β-内酰胺类与酶抑制剂联用或不同种类药物联用是防治鸡产酶大肠杆菌感染的有效措施之一.     

Bacteriological and molecular typing of Clostridium perfringens strains isolated in retail beef in Beijing,China

Clostridium perfringens is an important zoonotic pathogen. This study was designed to explore the prevalence and toxin types of C. perfringens in retail beef collected from Beijing, China. Among 221 beef samples collected, 53 samples were positive for C. perfringens, resulting in the average prevalence as 23.98%. By toxin gene-based typing, the most C. perfringens strains belong to type A (96.23%, 51/53), only 2 strains were identified as type D. By a multi-locus sequence typing (MLST)-based analysis, a total of 36 sequence types (STs) were detected, and the most STs (n=30) represented just a single strain. These finding suggested that the prevalence of C. perfringens in retail beef in Beijing was considerably high and these bacteria displayed extreme diversity in genetics.     

应用DPO-PCR方法特异性检测志贺氏菌

In this study, a dual-priming oligonucleotide (DPO)-based PCR method for specific detection of Shigella was established using ipaH gene of Shigella as the target gene. The results showed that detection sensitivity of the DPO-PCR method was 1.65×10² CFU/mL. Compared with conventional PCR primers, the DPO primers were easily designed, which simplified the design procedure of PCR primers. DPO primers were not sensitive to annealing temperature, which could efficiently amplify the target gene in the annealing temperature range from 50 to 70 ℃. Moreover, due to the special structure of DPO primers, it had a higher specificity than conventional PCR primers, and none nonspecific amplifications were produced in reaction. In the practical application, tests on 133 samples including frozen/fresh meat, fruits and vegetables, fresh milk, eggs and cooked food by the DPO-PCR method showed that 15 samples were Shigella positive, which were in accordance with the testing results using GB method (GB/T 4789.5-2012), showing good practicality and reliability. The DPO-PCR method provided a new tool for fast and accurate detection of Shigella.