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【目的】本研究选择原核表达系统表达鸭坦布苏病毒(Duck Tembusu virus, DTMUV)核衣壳蛋白(Capsid protein),并制备其多克隆抗体,为DTMUV分子机制研究奠定基础。【方法】根据DTMUV-201909株基因序列,运用一步克隆技术将Capsid基因克隆至表达载体pET-30a(+)中,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,利用IPTG进行诱导表达,通过SDS-PAGE和Western blotting鉴定重组蛋白;使用ISA206佐剂与纯化后的重组蛋白混合乳化后免疫BALB/c小鼠,以获得多克隆抗体。间接ELISA方法测定获得的多克隆抗体效价,并对多克隆抗体进行Western blotting和间接免疫荧光试验(IFA)验证。【结果】试验成功构建pET-30a-Capsid重组质粒,SDS-PAGE结果显示,表达的重组蛋白大小约为18 ku,主要以包涵体形式存在;Western blotting结果表明,该蛋白能与抗His标签鼠单克隆抗体发生特异性反应,具有良好反应原性。间接ELISA结果显示,制备的鼠抗Capisd蛋白多克隆抗体效价可达1... 相似文献
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通过大肠杆菌表达系统表达补体C9重组蛋白,并制备其多克隆抗体,用于检测沉积菌体表面的补体C9分子;方法:通过PCR以兔肝细胞cDNA文库为模板扩增获得补体C9部分基因,并将其克隆至原核表达载体pEASY.-Blunt E1,经测序鉴定成功获得重组质粒pBlunt-C9。将其转化至大肠杆菌感受态细胞BL21(DE3)中,经IPTG诱导,Ni-NTA亲和层析柱纯化后,获得重组蛋白。将纯化的重组蛋白免疫BALB/c雌鼠,制备抗C9的鼠源多克隆抗体。结果显示,ELISA测得鼠抗C9多克隆抗体效价为1∶12800,Western Blotting表明,制备的多克隆抗体可以特异性识别沉积在金黄色葡萄球菌和链球菌表面的C9分子,为进一步研究补体的生物学功能以及巩膜复合体(MAC)抗细菌感染机制奠定了基础。 相似文献
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本试验旨在制备兔抗鸡toll样受体7(chicken toll-like receptor 7, chTLR7)多克隆抗体，根据GenBank已发表的chTLR7序列设计引物，采用RT-PCR方法扩增chTLR7胞外区基因片段，并构建原核表达重组质粒，通过优化蛋白表达条件，用获得的融合蛋白免疫兔制备多克隆抗体，通过免疫印迹及间接免疫荧光试验鉴定制备的兔抗chTLR7多克隆抗体的特异性。结果显示，该片段长度为1 122 bp，与预测的片段大小、碱基序列一致。构建的原核表达重组质粒pGEX-6P-1-chTLR7经IPTG诱导后，表达出预期63 kDa的重组蛋白，免疫兔可产生特异性抗体。制备的兔抗chTLR7多克隆抗体具有良好的特异性，为后续实验研究chTLR7传导通路、免疫机制等相关研究提供有利工具。 相似文献
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为研究化脓隐秘杆菌致病机制及其病原学诊断方法,本研究克隆了编码化脓隐秘杆菌溶血素蛋白的plo基因,并构建重组表达质粒pET-plo,转化大肠杆菌Rosetta (DE3)感受态细胞中诱导表达.SDS-PAGE检测结果显示,表达的重组蛋白约为62 ku,western blot分析表明表达的重组蛋白可以与鼠抗化脓隐秘杆菌血清发生反应.采用重组蛋白免疫新西兰白兔制备的多克隆抗体效价达到1∶128 000,western blot和琼脂双扩散试验表明制备的多克隆抗体能够与天然PLO蛋白发生反应.溶血试验表明重组蛋白能够溶解红细胞,制备的多克隆抗体能有效中和重组蛋白的溶血活性. 相似文献
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《中国兽医学报》2020,(6)
试验旨在表达、纯化扩展莫尼茨绦虫VASA蛋白,并制备兔抗VASA多克隆抗体。根据扩展莫尼茨绦虫全基因核苷酸序列设计特异性引物,利用PCR方法扩增vasa基因片段;将扩增产物与原核表达载体pET-22b(+)连接,获得重组质粒pET-22b-VASA;经Amp抗性筛选阳性菌,双酶切、PCR测序鉴定后,将其转化大肠杆菌(DE3)感受态细胞中,并进行IPTG诱导表达;重组融合蛋白经镍柱纯化后,进行Western blot鉴定;将融合蛋白免疫兔,制备多克隆抗体。结果显示,重组质粒pET-22b-VASA构建正确,通过IPTG诱导获得大小约32 200的VASA重组融合蛋白;Western blot分析表明其与鼠抗His单克隆抗体呈阳性反应。纯化的VASA蛋白免疫兔获得了多克隆抗体,ELISA检测其效价为1∶128 000。本试验成功制备了具有免疫原性的VASA蛋白及其兔源多克隆抗体,为VASA蛋白生物学功能及该蛋白在绦虫体内的分布等研究奠定基础。 相似文献
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为体外表达牛源多杀性巴氏杆菌(Pasteurella multocida)PlpE基因及制备抗PlpE蛋白多克隆抗体,根据PlpE基因核酸序列设计了1对特异性引物,通过PCR扩增PlpE基因,构建重组质粒pET-32a-PlpE;摇菌提取质粒,进行双酶切鉴定,鉴定正确后将重组质粒pET-32a-PlpE转化至BL21(DE3)感受态细胞,加入IPTG进行诱导,对诱导蛋白进行SDS-PAGE和Western Blot鉴定;使用纯化蛋白PlpE免疫北京大耳白兔,制备多克隆抗体,并进行间接ELISA和Western Blot分析。结果显示,扩增的PlpE基因大小为1 050 bp,在原核细胞中诱导表达的PlpE蛋白约56 kDa。间接ELISA结果显示,所制备多克隆抗体效价可达1:512 000;Western Blot结果显示,所制备多克隆抗体反应性良好。PlpE蛋白的成功表达和多克隆抗体的成功制备,为下一步多杀性巴氏杆菌诊断方法的建立和疫苗研发奠定了基础。 相似文献
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[目的] 利用原核表达系统表达1型鹅星状病毒（Goose astrovirus 1,GAstV-1）结构蛋白ORF2,并制备其多克隆抗体。[方法] 根据GAstV-1 TZ03株基因序列,对ORF2基因序列进行大肠杆菌密码子偏爱性优化和合成,将其克隆至原核表达载体pET-30a（+）,构建重组质粒pET-ORF2,转化大肠杆菌BL21（DE3）感受态细胞,IPTG诱导表达重组蛋白,通过SDS-PAGE和Western blotting鉴定重组蛋白;将鉴定正确的重组蛋白纯化后免疫家兔制备多克隆抗体,以间接免疫荧光试验鉴定多克隆抗体的特异性。[结果] 酶切和测序结果显示,成功获得重组质粒pET-ORF2;SDS-PAGE结果显示,表达的重组蛋白分子质量约为70 ku,主要以包涵体形式存在;Western blotting结果表明,该蛋白能与抗His标签鼠单克隆抗体发生特异性反应。间接ELISA检测制备的兔抗ORF2蛋白多克隆抗体效价达1：128 000;间接免疫荧光试验结果显示,制备的多克隆抗体能与GAstV-1发生特异性反应。[结论] 原核表达的GAstV-1 ORF2重组蛋白具有良好的免疫原性,制备的兔源多克隆抗体具有较高的效价和特异性,为研制GAstV-1相关诊断试剂奠定了基础。 相似文献
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猪唾液酸黏附素氨基端结构域基因的克隆表达及其多克隆抗体制备 总被引：1，自引：1，他引：0
本试验旨在克隆出猪唾液酸黏附素的近氨基端结构域基因,进而构建原核表达载体并诱导表达重组蛋白,以制备多克隆抗体。试验中应用RT-PCR技术从健康仔猪肺巨噬细胞中克隆出猪唾液酸黏附素的近氨基端结构域的cDNA序列,并将其亚克隆到原核表达载体pET-32a中,构建重组表达质粒pET-Sn150,成功表达了分子质量大小约为43.1 ku的γ亚单位重组蛋白。将重组蛋白pET-Sn150免疫小鼠,制备获得鼠抗pET-Sn150重组蛋白多克隆抗体,将得到的重组蛋白多克隆抗体采用间接 ELISA 和Western blotting试验方法检测,ELISA测定多克隆抗体的血清效价为1∶12800;Western blotting试验结果证明,制备的鼠抗pET-Sn150多克隆抗体可以与重组蛋白进行特异性结合,从而证明重组蛋白具有较好的免疫原性。本试验克隆出猪唾液酸黏附素的近氨基端结构域基因并成功表达,为进一步研究其结构与功能奠定了基础。 相似文献
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为制备鸡源宿主限制因子SAMHD1(chSAMHD1)的多克隆抗体,本试验根据chSAMHD1基因序列设计引物,扩增部分chSAMHD1片段,构建p ET-chSAMHD1原核表达质粒,并将其转化至BL21(DE3)大肠杆菌,经IPTG诱导和镍柱亲和层析纯化获得重组chSAMHD1蛋白。将重组蛋白免疫BALB/c小鼠制备抗chSAMHD1多克隆抗体,通过Westernblot和IFA测定多克隆抗体的反应性。结果显示,chSAMHD1重组蛋白的相对分子质量约为75.0 k Da,与预期大小一致;重组蛋白以包涵体形式表达;所制备的chSAMHD1多克隆抗体效价达到1:512 000;IFA结果证实,本试验所制备多克隆抗体能够特异性识别DF-1细胞中过表达的chSAMHD1蛋白。以上结果表明,该研究成功制备了chSAMHD1蛋白多克隆抗体,从而为鸡源SAMHD1蛋白功能的深入研究奠定了基础。 相似文献
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为了制备鸡干扰素调节因子7(interferon regulatory fator 7,IRF7)抗体,本研究通过分离鸡的外周血淋巴细胞,抽提RNA并反转录获得鸡淋巴细胞c DNA,利用聚合酶链式反应扩增获得鸡IRF7基因(ch IRF7),将该基因克隆入原核表达载体p COLD-TF中,构建成p COLD-TF-ch IRF7原核表达质粒。将测序正确的表达质粒转化入大肠杆菌BL21(DE3)感受态中,经0.25 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)低温诱导20 h。SDS-PAGE结果显示,融合蛋白获得高效表达,大小为107 k Da。切胶免疫BALB/c小鼠,制备鼠抗鸡IRF7多克隆抗体。激光共聚焦、Western blot实验结果显示,该抗鸡IRF7多克隆抗体能与真核质粒表达的ch IRF7蛋白发生特异性反应。该抗体的成功制备为进一步研究鸡IRF7的表达调控机制奠定了基础。 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