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为构建缺失马立克氏病病毒(Mareks disease virus,MDV)Meq基因簇microRNAs(Meq-clustered miRNAs的突变株,本研究在vv MDV GX0101细菌人工染色体(bacterial artificial chromosome,BAC)克隆的基础上,采用Red/ET同源重组技术将Meq-clustered miRNAs的编码基因进行缺失突变,经PCR鉴定及序列分析证明Meq-cluster miRNAs序列成功缺失后,提取Δmeq-miRNAs BAC DNA,转染鸡胚成纤维细胞(CEF)进行病毒拯救,用SYBR GreenⅠqRT-PCR检测病毒体外增殖特性。结果表明成功拯救出缺失MDV Meq-clustered miRNAs基因的感染性BAC克隆株GX0101Δmeq-miRNAs,且该缺失株与亲本株GX0101BAC具有相似的增殖曲线,Meq-clustered miRNAs是MDV体外复制的非必需基因。MDV Meq-clustered miRNAs基因缺失感染性BAC克隆株的成功构建为进一步研究MDV Meq-clustered miRNAs在MDV致病和致肿瘤方面的作用奠定了基础。 相似文献
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《中国预防兽医学报》2018,(10)
为构建猪源伪狂犬病病毒(PRV)变异株AH02LA的细菌人工染色体(BAC),本研究将转移载体pHA2-pUC19-H1-H2的DNA与PRV AH02LA株基因组DNA共转染鸡胚成纤维细胞(CEF)进行同源重组,利用转移载体中的GFP为筛选标记,获得携带GFP基因的gE/gI基因缺失突变株PRV-AH02LA(gE-/gI-)-pHA2。提取纯化的该重组病毒DNA转化E.coli DH10B,对获得的阳性克隆命名为BAC~(PRV-G)。提取BAC~(PRV-G)的DNA,将含gE、gI和其上下游同源片段的DNA与BAC~(PRV-G)的DNA共转染CEF,获得gE、gZ基因恢复的病毒命名为PRVAH02LA~(Rec)。比较亲本株PRV AH02LA、基因缺失株和恢复株的生长动力学,结果表明,恢复株的生长特性与亲本株无显著差异。本研究成功构建了PRV变异株AH02LA的BAC,并证实其为全基因组的感染性克隆,为PRV变异株的致病机理研究和新型疫苗研制提供了重要的平台。 相似文献
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本试验旨在对缺失meq基因的马立克氏病病毒(MDV)感染性克隆基因组中细菌人工染色体(BAC)序列的自我敲除进行分子鉴定。将含有表达cre重组酶真核表达盒的SC9-1重组质粒通过转染鸡胚成纤维细胞(CEF)拯救SC9-1重组病毒。将SC9-1重组病毒在CEF细胞上进行连续传代培养,利用病毒表达的cre重组酶在传代过程中逐步敲除病毒的BAC序列。分别提取1~10代不同代次SC9-1重组病毒的DNA作为模板,利用BAC序列特异性引物对病毒基因组中的BAC进行PCR检测。利用针对BAC序列gpt基因的特异性检测引物,以MDV保守基因pp38作为内参,对不同代次病毒基因组中BAC序列进行荧光定量PCR的检测。利用PCR扩增SC9-1病毒敲除BAC序列后基因组中残留loxp位点两端序列,通过测序分析进一步验证BAC序列的敲除。利用BAC序列特异性引物对不同代次病毒基因组中BAC序列的检测结果显示,1~5代病毒DNA均能扩增出600 bp的特异性目的条带,证实病毒的基因组中含有BAC序列。6~10代病毒DNA均不能扩增出特异性目的条带,证实病毒基因组中没有BAC序列。荧光定量PCR结果显示,病毒基因组中的BAC序列随着病毒的传代逐渐减少,直至第6代已经完全检测不到BAC序列。在病毒BAC序列敲除的过程中,对每代病毒BAC序列敲除后残留loxp位点序列进行PCR测序鉴定,结果显示BAC序列敲除后基因组中仅留下一个loxp位点,序列同源性均在99.7%以上。结果表明,利用cre/loxp系统将病毒在CEF细胞上连续6代传代培养可将MDV meq基因缺失株SC9-1的BAC序列完全敲除掉,且同源重组酶敲除BAC序列具有高度一致性。 相似文献
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《畜牧兽医学报》2016,(10)
拟构建一种新型火鸡疱疹病毒(HVT)全基因组细菌人工染色体(BAC),通过同源重组方法将mini-F载体插入HVT基因组的糖蛋白C(glycoprotein C,gC)基因的等位位点得到mini-F重组HVT,提取重组病毒的DNA转入大肠杆菌DH10B细胞,再转入GS1783细胞获得BAC,拯救病毒获得mini-F重组病毒和gC恢复病毒后,与亲本病毒(HVT FC126)比较生长动力学和对鸡马立克病的免疫效力。结果:获得数个BAC阳性克隆,取其中一个克隆(BAC~(HVT-G))成功拯救出HVT mini-F重组病毒(HVT~(BAC-ΔgC)),并成功获得了gC恢复毒株(HVT~(BAC-gC-R))。生长特性和免疫效力试验表明HVT~(BAC-gC-R)与HVT FC126无显著差异,而HVT~(BAC-ΔgC)增殖能力和免疫效力均明显下降。成功构建了HVT全基因组的感染性BAC;HVT的gC基因是一个非必需基因,但对病毒的增殖能力和免疫效力有重要影响。 相似文献
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为构建表达鸡新城疫病毒(NDV)融合蛋白(F)的重组马立克氏病毒(MDV),本研究采用RT-PCR方法从NDV强毒株F48E9基因组中扩增出病毒的融合蛋白F基因,构建由CMV启动子和BGH polyA组成的2.7 kb F基因表达盒。将其插入带有黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因(gpt)和MDV US2同源臂的中间转移载体pUAB-gpt中获得重组MDV转移载体pUAB-gpt-wF。将该转移载体与MDV-814疫苗株感染的鸡胚成纤维细胞(CEF)总DNA共转染CEF,经同源重组及gpt选择系统筛选,获得带有F基因表达盒的重组MDV(rMDV814-wF)。其体外增殖与亲本病毒没有差异。经间接免疫荧光试验、PCR、Southern-blot及western blot等试验证明,重组病毒在CEF中传至13代以上仍稳定表达NDV的F蛋白。该重组病毒的构建为MDV活载体疫苗的筛选及应用奠定了基础。 相似文献
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猪瘟病毒低温诱变疫苗Thiverval株感染性克隆的构建及病毒拯救 总被引：3，自引：0，他引：3
采用高保真DNA聚合酶,分7个片段扩增猪瘟病毒(CSFV)Thiverval株全基因组序列,克隆至pMD18-T载体并测序.经适当的连接策略将各片段连接克隆至低拷贝载体质粒pAC/F101,同时对病毒基因组5'和3'末端进行修饰,构建出含有T7启动子、榔头状(HH)核酶基因、CSFV Thiverval全基因组、丁型肝炎病毒(HDV)核酶基因和T7终止子的重组质粒pAC/F101/T1-7.利用T7 RNA聚合酶将线性化重组质粒pAC/F101/T1-7体外转录成基因组RNA,再使用脂质体转染将体外转录RNA转染PK-15细胞.通过传代、RT-PCR、免疫过氧化物酶细胞单层试验鉴定,表明成功的从感染性克隆拯救出活病毒粒子.猪瘟病毒低温诱变疫苗"Thiverval"株感染性克隆的构建及病毒的成功拯救,为进一步研究CSFV疫苗株的致弱机制提供了重要工具. 相似文献
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《中国预防兽医学报》2020,(9)
禽网状内皮组织增生病(RE)是禽类一种重要免疫抑制性疾病,目前尚无有效的商品化疫苗预防该病。为研制表达RE病毒(REV)gp90蛋白的重组鸡马立克氏病病毒(MDV),本研究构建了含有REV保护性抗原基因gp90的pCAGGS-gp90表达质粒,将gp90表达框架克隆入pENTR1载体,获得重组质粒pENTR1-gp90。通过pENTR1-gp90与p814-5US2KanccdB重组粘粒的LR反应,将gp90表达框架插入p814-5粘粒中MDV 814疫苗株基因组US2基因内,构建重组粘粒p814-5-gp90。将该重组粘粒转染鸡胚成纤维细胞(CEF),获得重组MDV r814-gp90株。将该重组病毒在CEF中连续传至20代后对第5、10、15、20代病毒经PCR扩增并测序鉴定;利用间接免疫荧光试验(IFA)和western blot检测重组MDV感染细胞中gp90蛋白的表达;绘制重组MDV的生长曲线,分析其体外复制特性。结果显示,正确构建了含有gp90表达框架的重组粘粒p814-5-gp90。将该重组粘粒转染CEF后能够引起典型的蚀斑病变,且蚀斑形态大小与亲本病毒814株无明显差异。PCR及测序鉴定结果显示,重组病毒r814-gp90基因组中正确插入gp90基因表达框架,且其能够在该重组病毒中稳定存在。IFA及western blot检测结果表明,r814-gp90株能够在感染的CEF中表达gp90蛋白。体外生长曲线显示,r814-gp90株在CEF中的复制能力与亲本病毒无明显差异。以上结果表明,本研究正确构建了表达REV保护性抗原gp90基因的重组MDV,为研制RE重组MDV活载体疫苗奠定了基础,对RE以及REV与MDV混合感染的防控具有重要意义。 相似文献
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采用聚合酶链反应或反转录聚合酶链反应扩增出H5亚型禽流感病毒(AIV)的HA基因、网状内皮增生症病毒的长末端重复序列(LTR)、马立克氏病病毒(MDV)Rispens CVI988毒株基因组的sorf 1和sorf 2序列、两端带loxp位点的lac/smGFP标志基因,构建含这些基因的转移载体质粒pMHA;以MDV Rispens CVI988毒株的基因组DNA和PMHA质粒DNA共转染鸡胚成纤维细胞(CEF),采用同源重组方法将LTR、lac/smGFP和HA基因插入到MDV基因组,获得重组病毒rMDV-HA/GFP;以cre介导的同源重组去除lac/smGFP标志基因,再转染CEF,获得仅带LTR启动子和HA基因的重组MDV疫苗毒株rMDV-HA.rMDV-HA仍保留了MDV RispensCVI988疫苗毒株的复制特点,并能稳定表达AIV的HA. 相似文献
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本实验以独立启动子控制的增强型绿色荧光基因(GFP)作为报告基因,同时将CMV启动子及其多克隆位点与之连接,构成外源基因表达盒,插入到马立克病毒(MDV)复制非必需区基因(短独特区US2等)构成的同源臂中,构建成重组马立克病毒的通用载体。鉴定正确后,将转移载体与提取的MDV基因组共转染鸡胚成纤维细胞(CEF),同源重组获得具有感染性的重组病毒,待病毒蚀斑出现后,荧光显微镜下观察,可见到明显的绿色荧光病毒蚀斑,经三次筛选,初步分离到重组病毒。结果表明,转移载体与MDV基因组共转染可获得感染性病毒,US2基因可作为重组病毒构建中的外源基因插入位点,证实通用转移载体的构建是可行的,为重组马立克病毒新型疫苗的研究奠定物质基础。 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