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对旋毛虫新生幼虫 期特异性基因进行筛选与鉴定,结果表明:对新生幼虫cDNA文库进行核酸杂交筛选，获得7个阳性克隆，其大小在1.4 kb左右；用放射性同位素α32P标记cDNA探针后，与旋毛虫成虫、肌幼虫、新生幼虫、成虫/新生幼虫cDNA文库质粒DNA进行Southern印迹杂交，结果与成虫/新生幼虫、新生幼虫cDNA文库质粒DNA有杂交而与成虫、肌幼虫cDNA文库质粒DNA不杂交，因此该探针是新生幼虫期所特有的cDNA片段。 相似文献
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旋毛虫新生幼虫差减cDNA文库的构建及其步筛选 总被引：9，自引：6，他引：3
利用差减杂交（减法杂交，subtractive hybridization)技术，以旋毛虫新生幼虫（newborn larvae,NBL)的cDNA作为试验方（tester)，以肌幼虫＋成虫的cDNA作为驱动方（driver)，制备新生幼虫差减cDNA,利用T载体构建NBL差减cDNA文库，获克隆株90个，以试验方的差减cDNA第2次PCR产物＋未差减cDNA为试验方探针，以驱动方的差减cDNA第2次PCR产物＋未差减cDNA为驱动方探针，在NBL差减cDNA文库中初筛NBL的期特异性克隆，获初筛克隆24个。这些初筛斯异性克隆进一步用Southern blot确认鉴定，获直阳性期特异性克隆2个（NBL SSC1，NBL SSC2）.DNASIS以及Blaster的分析结果显示，这2个克隆为旋毛虫的2个新基因，其中NBL SSC1编码糖蛋白，NBL SSC2编码丝氨本蛋白酶，本试验为旋毛虫期特异性基困全长序列的调取，分析，鉴定以及强保护性抗原基因的筛选奠定了基础。 相似文献
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旋毛虫5日龄成虫期特异性基因片段的克隆及鉴定 总被引：5，自引：1，他引：4
利用差减抑制杂交（SSH）技术，以旋毛虫5日龄成虫的cDNA为试验方（tester），以肌幼虫＋3日龄成虫的cDNA为驱动方（driver），制备5日龄成虫差减cDNA，并与pT-Adv载体相连接，转入大肠埃希氏菌TOP 10F。以试验方的差减cDNA PCR产物＋未差减cDNA为试验方探针，以驱动方的差减cDNA PCR产物＋未差减cDNA为驱动方探针，对5日龄成虫差减cDNA克隆进行初步筛选，对筛选到的阳性克隆进一步用southern bolt进行鉴定，并对其测序，进行序列分析。结果表明，获得1个大小为464bp的旋毛虫5日龄成虫期特异性cDNA片段，经BLAST软件分析表明，该序列是1个未见报道的旋毛虫基因序列片段，可能编码1种表皮胶原蛋白。这为旋毛虫5日龄成虫期特异性基因全长序列的钓取、分析及鉴定奠定了基础。 相似文献
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旋毛虫肠道1期幼虫抗原性基因的筛选与分析 总被引：1，自引：1，他引：0
筛选旋毛虫肠道1期幼虫抗原性基因。利用λZAP载体构建旋毛虫肠道1期幼虫cDNA文库。用感染旋毛虫26 d猪血清对其进行免疫学筛选,对筛选出的阳性克隆进行测序与分析;应用RT-PCR方法对编码p46 000蛋白强阳性克隆基因在不同发育时期虫体转录情况进行检测。成功构建了旋毛虫肠道1期幼虫cDNA文库,其库容量为1.5×106pfu,重组率为95%,插入片段在400 bp~2 000 bp之间,扩增后文库滴度为1.5×1012pfu/mL。筛选出阳性克隆33个,序列分析表明,有20个克隆为同一已知基因,称为旋毛虫p46 000蛋白,编码旋毛虫半胱氨酸蛋白酶抑制剂,且在筛选过程中反应信号均较强;还发现了一些旋毛虫新基因,如编码Ubc蛋白,GM13181蛋白,Rieske硫铁蛋白1等。本研究获得了一个高丰度、反应原性较强抗原基因,其编码蛋白为半胱氨酸蛋白酶抑制剂。 相似文献
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中国旋毛虫分离株成虫和新生幼虫基因文库的构建及筛选 总被引：21，自引：5，他引：16
利用λＺＡＰⅡ载体构建了中国分离株Ｔｒｉｃｈｉｎｅｌａｓｐｉｒａｌｉｓ及Ｔｒｉｃｈｉｎｅｌａｎａｔｉｖａ成虫和新生幼虫ｃＤ－ＮＡ基因文库,检测结果表明,所克隆的ｃＤＮＡ片段分布在０．２～２．０ｋｂ之间,表明２个ｃＤＮＡ基因文库对ｍＲＮＡ的覆盖面很大。利用ＲＡＰＤ技术,以肌幼虫基因文库作对照,采用７０个单引物及２０对复合引物对以上２个文库进行筛选扩增,结果：（１）共获得２条Ｔ．ｓｐｉｒａｌｉｓ及４条Ｔ．ｎａｔｉｖａ的成虫与新生幼虫特异性基因片段,鉴于成虫与新生幼虫是旋毛虫的２个侵袭性阶段,其所特有的特异性基因片段极有可能就是这２个时期虫体的侵袭性因子基因,即所要寻找的强保护性抗原基因,从而为后续研究奠定了基础;（２）发现１条Ｔ．ｓｐｉｒａｌｉｓ种特有的基因片段,这一特异性基因片段为虫种鉴定提供了物质条件;（３）发现旋毛虫同种不同分离株（中国分离株及法国分离株）间ｃＤＮＡ文库扩增图谱完全相同,进一步证实了旋毛虫种内基因的低流动性 相似文献
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构建猪肌生成抑制素(MSTN)成熟蛋白编码序列的克隆与表达载体，并对mRNA进行转录水平的检测，为获得较好的猪肌生成抑制素抗原、制备抗体打下良好的基础。猪MSTN基因的cDNA去除信号肽后，用PCR扩增的1．2kb目的片段与pMDl8-T载体连接，将克隆载体的质粒DNA与同样用BamH Ⅰ和Sal Ⅰ限制性内切酶酶解的表达载体pET28a( )质粒定向连接，对筛选出的阳性克隆用酶切及测序法鉴定。对猪MSTN基因成熟蛋白编码序列进行反转录，对mRNA Northern杂交进行转录水平的检测。结果，所得到的序列与所设计的序列完全一致，表明成功地进行了猪MSTN基因编码序列的克隆及筛选；目的片段定向插入，阅读框架正确；RT-PCR成功地反转录得到约1．2kb的目的DNA片段，Northern杂交后对mRNA印迹放射自显影，见到清晰的1．2kb特异带。 相似文献
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猪带绦虫六钩蚴cDNA文库的构建与筛选 总被引：1，自引：0，他引：1
在体外将猪带绦虫虫卵孵化为有活力的六钩蚴。采用商品化试剂盒抽提六钩蚴总 RNA、m RNA,反转录为双链c DNA,再加 Eco R Adapter。在去除小片段后 ,dsc DNA与 λgt11噬菌体 DNA连接 ,经蛋白包装 ,构建了猪带绦虫六钩蚴λgt11c DNA文库。该文库效价为 3.5× 10 9pfu/ m L ,重组 DNA片段大小为 0 .5～ 3.2 kb,平均为 1.6 kb,蓝白斑比 1∶ 9。用抗体探针对文库进行免疫学筛选 ,在消除非特异性反应的基础上筛选约 10 6 重组子 ,共得到 118株强阳性克隆 ;应用 PCR鉴定上述部分阳性克隆 ,均扩增到 0 .5 kb以上的片段。结果显示 ,构建的文库合格 ,含六钩蚴所有抗原基因 ,可用于六钩蚴 c DNA克隆的筛选 ;用免疫学与 PCR联合筛选 c DNA文库 ,可消除假阳性 相似文献
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猪肺炎支原体诱导家蝇幼虫抑制性消减文库的构建 总被引：2，自引：0，他引：2
采用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建猪肺炎支原体(Mycoplasma hyopneumonia,Mhp)诱导家蝇幼虫后产生差异表达基因的消减cDNA文库,并利用反向Northern杂交技术获得差异基因.结果表明,随机挑取的阳性克隆的大小均为200～750 bp,通过斑点杂交技术共筛选出186个差异基因片段,对其克隆、测序及同源性分析后鉴定出20种编码蛋白的基因片段和21个无同源序列.家蝇幼虫经猪肺炎支原体诱导后产生与抗病原体相关的基因及大量的未知基因. 相似文献
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从纯化的RHDV中提取电泳纯核酸,以此为模板在DNA聚合酶催化下,加六核苷酸随机引物和dATP、dCTP、dGTP及Digll-dUTP进行标记,制成DIG探针。用此探针与RHDV核酸作斑点杂交和Southern杂交,均显强阳性反应;与从RHDV-mRNA构建的RHDV cDNA文库中的2.2kb质粒DNA亦显阳性反应。用同法标记2.2kb质粒DNA,不仅与同 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