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采用PCR方法扩增驽巴贝虫吉林分离株BC-48基因片段，将扩增产砌与pGEM—TEasy载体连接，重组质粒经PCR、单酶切鉴定后测序；构建BC-48的重组pGEX-4T-2表达载体．经IPTG诱导表达后，进行SDS-PAGE、Western—blotting分析。结果显示，克隆的BC-48基因片段长610bp，含有一个570bp的开放阅读框，编码189个氨基酸，与GenBank中USDA株（U46551）的同源性为96．7％；表达的融合蛋白为45ku，能被驽巴贝虫阳性血清识别；表明该融合蛋白具有较好的反应原性。 相似文献
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构建牛瑟氏泰勒虫P23基因片段原核重组表达质粒,表达重组蛋白。采用PCR方法扩增牛瑟氏泰勒虫P23表面蛋白基因,将扩增产物与pMD18-T-Simple载体连接,测序,验证扩增产物。将P23基因片段克隆到载体pGEX-4T-3上,经酶切分析、PCR鉴定后,IPTG诱导表达,最后用SDS-PAGE和Western blotting分析鉴定表达产物。结果表明克隆的P23基因片段为684 bp,重组质粒pGEX-4T-3/P23构建成功;SDS-PAGE显示目的蛋白相对分子质量约为58 ku;Western blotting分析结果显示,与牛瑟氏泰勒虫阳性血清发生反应,而与牛瑟氏泰勒虫阴性血清无反应,表明牛瑟氏泰勒虫P23表面蛋白具有良好的抗原性和特异性,提示可以利用融合蛋白来建立检测抗体的间接ELISA诊断方法。 相似文献
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应用PCR方法扩增了牛瑟氏泰勒虫吉林株P33表面蛋白基因片段,并将扩增产物与pMD18-T载体连接,重组质粒经PCR、双酶切鉴定后测序;构建P33重组pGEX-4T-3表达载体,转化大肠杆菌BL-21,经IPTG诱导表达后.进行SDS-PAGE、免疫印记分析.结果显示,获得P33基因完整开放阅读框长868 bp,编码292个氨基酸,与中国株同源性为99.1%;表达的融合蛋白相对分子质量为59 000,能被牛瑟氏泰勒虫阳性血清识别,表明该融合蛋白具有较好的免疫原性. 相似文献
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根据环形泰勒虫TaA1272-1株裂殖体表面抗原(TaSP)基因的序列设计了1对引物，用PCR扩增出该基因长为393bp的高免疫原性区片段(SBxp)。将扩增产物连接到pGEM-T Easy载体上，转化入大肠埃希氏菌JM109中进行克隆。随后将重组质粒pGEM-SBxp和表达载体pGEX-4T-1分别以BamHⅠ、XhoⅠ酶切后，将酶切的目的片段SBxp亚克隆到原核表达载体中构建了重组表达载体pGEX-4T-1-SBxp，并将其转化到BL21宿主菌中。提取重组质粒经BamHⅠ、XhoⅠ酶切、PCR鉴定和测序确证后，阳性克隆用IPTG诱导表达。收集不同时间的菌液进行SDS-PAGE电泳和Western blotting检测。结果，SBxp基因在大肠埃希氏菌中成功表达，其表达产物为分子质量43ku的融合蛋白，该产物能被牛环形泰勒虫阳性血清所识别。 相似文献
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利用PCR方法扩增产单核细胞李斯特菌（LM）的溶血素基因hlyA,获得一条大小约为1600bp的目的片段,将其与pMD18-T载体连接,经酶切、PCR鉴定,命名为pMD18-T—hlyA。测序结果显示所扩增的hlyA基因与GenBank中发表的hlyA的序列同源性为99％。利用含有BamHⅠ／XhoⅠ酶切位点引物B从pMD18-T—hlyA扩增不含信号肽序列的目的片段B,获得了大小约为1500bp的基因片段,该片段与pET32a表达载体经BamHⅠ／XhoⅠ处理后进行连接,转化BL21受菌体。通过酶切、PCR鉴定及序列测定后获得阳性pET32a-hlyA表达重组质粒,将重组菌用终浓度为0．1mmol／L的IPTG进行诱导表达,结果表明重组菌在诱导2h～3h表达量最高,融合蛋白约占菌体的40％,分子量大小约为80ku;利用LM的阳性血清进行Western blot分析,证实该融合蛋白可以被LM阳性血清所识别,为今后研制针对该蛋白的单克隆抗体和建立间接ELISA诊断方法提供了条件。 相似文献
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PRRSV NC株ORF3基因的克隆、序列分析及表达 总被引：1，自引：0，他引：1
为原核表达猪繁殖与呼吸综合征病毒(PRRSV)的ORF3基因,本研究根据GenBank登录PRRSV美洲株ATCC VR2332的ORF3基因序列,利用Primer 6.0软件设计合成一对特异性引物,经RT-PCR扩增得到了大小为765bp的片段。将扩增的ORF3基因截短为495bp和750bp片段分别克隆于原核表达载体pGEX-KG中,在IPTG的诱导下进行Gp3重组蛋白的截短表达,经western blot检测证实表达的2种截短重组蛋白均具有良好的与抗体的反应活性,从而为进一步研制新型疫苗提供了实验依据。 相似文献
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采用PCR方法扩增吉林延边地区猪附红细胞体A1基因片段,将扩增产物与pMD18-T载体连接,重组质粒经PCR、酶切鉴定后测序;分别构建A1重组pGEX-4T-1和PET-28-a表达质粒,经IPTG诱导表达后,进行SDS-PAGE、Western blot分析。结果显示:克隆的A1基因片段长1 044 bp,含有1个999 bp的开放阅读框,编码333个氨基酸,与GenBank中A1基因序列(AM265536)的同源性为97%;表达的融合蛋白分子量分别为64 ku和40 ku,能被猪附红细胞体阳性血清识别。表明该融合蛋白具有较好的免疫反应活性。 相似文献
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根据GenBank(D12692.1)上发表的牛瑟氏泰勒虫HSP70基因序列设计、合成1对特异性的引物,采用PCR方法扩增牛瑟氏泰勒虫HSP70基因片段,将扩增产物与pMD18-Tsimple载体连接,重组质粒经PCR、酶切鉴定后测序;用pGEX-4T-1表达载体构建重组质粒pGEX-HSP70,经IPTG诱导表达后进行SDS-PAGE、Western-blot分析。结果表明:扩增的HSP70基因与D12692.1序列同源性为99.3%;表达的融合蛋白分子质量约为68ku,而且可与牛瑟氏泰勒虫阳性血清发生特异性反应。 相似文献
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牛瑟氏泰勒虫P33表面蛋白基因的克隆与序列分析 总被引：3，自引：0，他引：3
为分析吉林省流行的牛瑟氏泰勒虫基因序列，根据GenBank上发表的牛瑟氏泰勒虫P33表面蛋白基因序列设计合成一对引物，用PCR方法扩增出牛瑟氏泰勒虫的基因片段，并成功地将该基因纯化后克隆到pGEM-TEasy载体上，将经EcoRⅠ酶切鉴定和PCR鉴定为阳性的重组质粒进行测序。结果表明克隆的基因片段长度为868bp，编码283个氨基酸，有2个潜在的糖基化位点。核苷酸同源性分析表明，该基因片段与韩国株（AF521557）、日本株（AB016280）、俄罗斯株（AB016279）的核苷酸序列同源性分别为99．4％、88.0％、88．1％。 相似文献
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魏静 《四川畜牧兽医学院学报》2009,(4):28-32
在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。 相似文献
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本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。 相似文献
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以国际标准强毒Ｒ株人工感染非免疫产蛋鸡,定时扑杀,分别从鼻窦、眶下孔、气管、肺、气囊、卵巢和输卵管分离ＭＧ,并收集感染鸡所产蛋分离ＭＧ。结果表明,人工感染４８小时后上、下呼吸道及肺已被全面感染,９６小时气囊已被感染,１２０小时输卵管已能分离到ＭＧ,卵巢始终分离不到ＭＧ。人工感染鸡自１４４小时便能在其所产蛋中分离出ＭＧ。药物治疗能在７２小时内消除感染,油乳剂苗则需２４天后逐渐降低蛋内ＭＧ分离率,药物卵内注射、种蛋药浴、高温处理均能杀死卵内ＭＧ,但以研制的种蛋浸泡剂药浴效果为最好。 相似文献
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REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air. 相似文献
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Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF
Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo. 相似文献