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为研究绵羊肺腺瘤病病毒(JSRV)表面蛋白(SU)的致瘤机制,本研究构建稳定表达SU的羊肺细胞A549细胞系.采用PCR方法从含su基因的pGEX-4T-1-SU重组质粒中扩增SU编码序列,将其克隆至真核表达载体pcDNA3.1(+)中,转染A549细胞.通过G418筛选,对转染阳性细胞进行纯化,获得稳定表达JSRV SU蛋白的A549细胞系.以间接免疫荧光及western blot鉴定SU的表达状况,并运用共聚焦显微镜确认SU蛋白的亚细胞定位.结果表明,重组蛋白SU在A549细胞中有效表达,而且主要分布于细胞质中.该细胞系的建立为SU生物学功能的体外研究提供了平台. 相似文献
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为了建立原核高效表达体系,从自然感染绵羊肺腺瘤病毒内蒙毒株的肺肿瘤组织中提取基因组DNA,应用PCR技术分别扩增编码囊膜蛋白(env)基因的表面蛋白(surfaceprotein,su)和跨膜蛋白(transmembrane protein,TM)区域基因,通过T—A克隆的方法分别克隆入pGEM—TEasy载体中,然后利用设计的起始密码、终止密码以及相应酶切位点的引物,把env基因的SU区和TM区基因亚克隆到表达载体pGEX-4T~1中,分别构建SU和TM的重组表达质粒,经酶切、PCR及DNA测序鉴定后,将阳性质粒转化入大肠杆菌BL21CodonPlus中并在IPTG的诱导下表达,表达产物用SDS—PAGE分析鉴定。结果表明,该基因可以在大肠杆菌中以稳定的包涵体形式高效表达,表达的SU和TM融合蛋白表观分子质量约为68ku和46ku,表达量分别占全菌蛋白的25%和30%,并且从包涵体分离纯化得到的表达蛋白具有较高纯度,是一种理想的免疫原。 相似文献
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绵羊肺腺瘤病毒内蒙毒株囊膜蛋白基因在大肠杆菌中的分段表达 总被引:2,自引:0,他引:2
为了建立原核高效表达体系,从自然感染绵羊肺腺瘤病毒内蒙毒株的肺肿瘤组织中提取基因组DNA,应用PCR技术分别扩增编码囊膜蛋白(env)基因的表面蛋白(surface protein,SU)和跨膜蛋白(transmembrane protein,TM)区域基因,通过T-A克隆的方法分别克隆入pGEM-TEasy载体中,然后利用设计的起始密码、终止密码以及相应酶切位点的引物,把env基因的SU区和TM区基因亚克隆到表达载体pGEX-4T-1中,分别构建SU和TM的重组表达质粒,经酶切、PCR及DNA测序鉴定后,将阳性质粒转化入大肠杆菌BL21 CodonPlus中并在IPTG的诱导下表达,表达产物用SDS-PAGE分析鉴定。结果表明,该基因可以在大肠杆菌中以稳定的包涵体形式高效表达,表达的SU和TM融合蛋白表观分子质量约为68 ku和46 ku,表达量分别占全菌蛋白的25%和30%,并且从包涵体分离纯化得到的表达蛋白具有较高纯度,是一种理想的免疫原。 相似文献
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绵羊肺腺瘤病毒NM株囊膜基因的定向克隆与融合表达 总被引:1,自引:0,他引:1
根据绵羊肺腺瘤病毒(SJRV)NM株基因组全序列,设计了1对包含囊膜(env)基因在内的引物,并在5′端分别设计了HindⅢ和BnmHⅠ酶切位点,利用PCR技术扩增env基因,通过连接反应将其克隆于pET-30b表达载体上。序列分析表明,env基因全长1836bp,编码612个氨基酸残基;构建表达重组质粒env—pET—30b,转化大肠埃希氏菌BL21,经IPTG于37℃诱导培养,SDS—PAGE电泳分析表明,表达的env基因融合蛋白分子质量约为69ku。 相似文献
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以猪伪狂犬病病毒(PRV)Min-A株pMD-gE质粒为模板,利用所合成的特异性引物进行PCR扩增,获得了1 000 bp的DNA片段,并将其克隆至表达载体pET-30a中,转化到大肠杆菌中进行了原核表达,对表达产物进行了抗原性分析;在此基础上,用纯化的gE蛋白免疫BALB/c小鼠,筛选和鉴定了分泌抗gE蛋白单抗的杂交瘤细胞株。结果显示,原核表达的gE蛋白具有良好的抗原性,其分子质量为46 ku。筛选获得3株分泌抗gE蛋白单抗的杂交瘤细胞株,Western-blot和间接免疫荧光试验证实,所获得的3株单抗具有良好的特异免疫反应原性。3株单抗均为IgG1亚类,且轻链均为κ链。 相似文献
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用大肠杆菌BL21表达了马立克氏病病毒(MDV)强毒GA株的囊膜糖蛋白B(gB)基因,通过SDS-PAGE电泳分离表达蛋白条带,切下并碾碎后作为免疫原制备单克隆抗体。通过ELISA和间接免疫荧光试验(IFA),得到1株阳性单克隆抗体杂交瘤细胞株7C8。该单抗腹水的ELISA效价为1:2^12,IFA效价为1:800,与MDV不同致病型毒株CVI988、GA、RB1B、MD11、648A株感染的鸡胚成纤维细胞(CEF)均呈IFA阳性。1:100稀释时与GA株感染的CEF在斑点酶联免疫吸附试验(dot-ELISA)中呈阳性。 相似文献
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为制备猪圆环病毒4型(PCV4)Cap蛋白单克隆抗体,以原核表达的重组Cap蛋白免疫6~8周龄小鼠,三次免疫后取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合。利用亚克隆技术和间接免疫荧光(IFA)方法筛选到2株阳性单克隆杂交瘤细胞株,分别命名为5H9和2F3。间接免疫荧光和Western blot试验表明,2株单抗均能特异性识别293T细胞中特异表达的Cap蛋白,且5H9和2F3的IFA效价分别为1∶12 800和1∶6 400。亚类鉴定结果表明,两株单抗重链均属于IgG1,轻链类型为kappa型。本研究制备的抗PCV4 Cap特异性单抗,为PCV4检测方法的建立和流行病学调查提供了有效的生物材料,也为该病毒的分离鉴定与Cap蛋白功能的探究奠定了基础。 相似文献
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《中国兽医杂志》2016,(12)
传染性囊病病毒(Infectious Bursal Disease Virus,IBDV)的非结构蛋白VP5在病毒感染细胞的不同阶段发挥着抑制或诱导细胞凋亡的重要作用。为制备抗血清I型IBDV VP5蛋白的单克隆抗体,取经His-VP5蛋白免疫4次的BALB/c小鼠的脾细胞与骨髓瘤细胞(SP2/0)进行融合,3次亚克隆后获得2株能稳定分泌VP5蛋白单克隆抗体的杂交瘤细胞株,分别命名为5EC7和2BE5。经间接ELISA测定,以上2株单抗的亲和力解离常数分别为1.14×10~(-10)和4.87×10~(-10),亚类分别为Ig G2a和Ig G1。通过Western Blot和间接免疫荧光试验检测,2株单抗均能识别IBDV感染DF-1细胞后产生的VP5蛋白。Western Blot试验表明,2株单抗识别的抗原表位区域为VP5 N-端的1~10 aa。该单抗为研究血清I型IBDV VP5蛋白的生物学功能及病毒致病机理奠定基础。 相似文献
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《中国兽医杂志》2015,(4)
为制备CIAV VP2单克隆抗体,将原核表达的His-VP2融合蛋白作为免疫原免疫BALB/c小鼠。经4次免疫后,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合。将原核表达的GST-VP2融合蛋白作为包被抗原,利用间接ELISA筛选阳性克隆。阳性细胞株经3次亚克隆后,获得3株稳定分泌抗CIAV VP2蛋白的杂交瘤细胞克隆,分别命名为3D7、3B8、2D3。经间接ELISA测定,以上3株杂交瘤细胞腹水效价分别为1:8.0×10~5、1:4.0×10~5,以及1:6.4×10~6,亲和力解离常数分别为7.34×10~(-11)、2.65×10~(-11),以及2.98×10~(-11)。IgG亚类分别为IgG1、IgG2b以及IgG2b。经Western Blot试验证明,3D7株单抗能特异地识别CIAV VP2蛋白,其识别的抗原表位区域为VP2 N-端的20~40 aa。经IFA试验证明,3株单抗均能识别天然的CIAVVP2蛋白。此单抗为研究CIAVVP2的生物学功能和CIAV致病机理奠定基础。 相似文献
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猪繁殖与呼吸综合征病毒核衣壳蛋白的原核表达及其单克隆抗体的制备 总被引:1,自引:1,他引:0
试验通过RT-PCR法扩增出猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)核衣壳蛋白(N蛋白)基因并克隆到原核表达载体pet-30a中,转入到BL21感受态细胞,经IPTG诱导表达,使用SDS-PAGE和Western blotting分析表达产物的特性。利用Ni柱纯化该重组蛋白,采用NC膜皮下包埋法免疫Balb/C小鼠,取免疫后的脾细胞与SP2/0细胞进行融合后筛选杂交瘤细胞株,检测杂交瘤细胞株的特性并制备单克隆抗体。SDS-PAGE和Western blotting结果表明,该重组蛋白表达正确,约为20 ku,能被抗PRRSV的阳性血清特异性识别。超声后用Ni柱纯化,经SDS-PAGE分析可得单一的目的条带。NC膜皮下包埋法免疫效果良好,通过细胞融合、ELISA筛选获得3株能稳定分泌抗PRRSV N蛋白抗体的杂交瘤细胞株(H7、F7、C8),制备出高特异性的针对N蛋白的H7单抗。IFA与Western blotting结果显示制备的单抗可与病毒N蛋白产生特异性反应。亚型鉴定为IgG2b型,染色体分析证实杂交瘤细胞染色体数目正确。结果成功实现了N蛋白的原核表达,并获得高特异性的单克隆抗体,为PRRSV N蛋白抗原的检测奠定基础。 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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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
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乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制. 相似文献