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应用PCR方法扩增出亚洲Ⅰ型口蹄疫病毒的VP1基因，继而构建出真核表达载体pVAX—VP1。再将含有巨细胞启动子CMV的目的基因表达盒连接到穿梭载体pVAX△E3上，筛选出VPl表达盒方向与E3区转录方向一致的重组子，获得转移载体。再经酶切，连接等方法获得重组腺病毒质粒。利用脂质体介导将重组质粒转染DK细胞，转染3次后，细胞出现明显的腺病毒病变。经PCR扩增、测序检测及基因组酶切鉴定，证明该病毒即为重组犬2型腺病毒。命名为CAV2／FMDV。经验证，该重组毒可稳定遗传，其毒价为10^3.5TCID50／mL。 相似文献
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为构建猪附红细胞体ENO基因重组腺病毒穿梭质粒,试验根据GenBank中登录的猪附红细胞体ENO基因序列（登录号：CP002525.1）设计特异性引物,对ENO基因进行PCR扩增,并将纯化后的PCR产物克隆到pMD19-T载体中。用Kpn Ⅰ和Xho Ⅰ对pMD19T-ENO进行双酶切后,将其亚克隆至腺病毒穿梭载体PCR259中,构建PCR259-ENO重组质粒,提取重组质粒进行鉴定。应用脂质体介导转染法将鉴定正确的PCR259-ENO重组穿梭质粒转染293细胞,应用间接免疫荧光法（IFTA）检测ENO基因在293细胞中的表达。结果显示,试验克隆的ENO基因长为1 182 bp,编码393个氨基酸,与GenBank中ENO基因序列（登录号：CP002525.1）同源性为99%。构建的重组腺病毒穿梭质粒PCR259-ENO经PCR和酶切鉴定正确,并且能在293细胞中表达,表明ENO基因成功插入腺病毒穿梭质粒PCR259中,重组腺病毒穿梭质粒PCR259-ENO构建成功。 相似文献
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《动物医学进展》2015,(7)
构建以多层纳米颗粒为载体的O型口蹄疫病毒基因重组疫苗。在GenBank中查询编码O型口蹄疫病毒(FMDV)VP1 141-160氨基酸和VP2 1-33氨基酸的核苷酸序列,合成该两段基因的重组基因并命名为VP1-VP2基因。再通过PCR扩增VP1和VP2基因,并将VP1、VP2及VP1-VP2基因分别插入到真核表达载体pcDNA3.1中,构建3种重组真核表达质粒(pcDNA3.1-VP1、pcDNA3.1-VP2和pcDNA3.1-VP1-VP2),并分别对重组质粒进行酶切、PCR鉴定及测序分析。将构建的重组质粒制备成多层磷酸钙纳米颗粒,检测其粒径、稳定性及体外转染效率。重组质粒酶切结果显示,基因片段大小与预期相符;测序显示与GenBank中相应基因序列同源性为100%。制备的多层纳米颗粒粒径约为530nm;在4℃过夜保存后,溶液均匀透明;体外转染HEK293细胞的转染效率在9.6%左右。成功构建了以多层磷酸钙纳米颗粒为载体的O型口蹄疫病毒VP1和VP2基因重组质粒,为进一步研究该基因疫苗奠定了基础。 相似文献
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《畜牧与兽医》2015,(11):1-5
为研究内溶素SAL-2和人溶菌酶h LYZ的联合抑菌作用,本研究通过PCR方法分别从携带SAL-2和h LYZ基因的质粒中扩增目的片段,连接至腺病毒穿梭载体的多克隆位点上,构建重组穿梭载体p Shuttle-SAL和p Shuttle-LYZ。成功构建的重组穿梭质粒与Ad-easy骨架质粒在BJ5183中发生同源重组,重组得到的阳性重组腺病毒载体在脂质体介导下转染HEK293细胞,包装产生重组腺病毒Ad-SAL、Ad-LYZ、Ad-SALLYZ和对照重组腺病毒Ad-GFP,通过RT-PCR和Western blot方法检测mR NA和蛋白表达后,将构建的重组腺病毒进行扩增纯化并测定病毒滴度。结果表明,成功构建的重组腺病毒感染细胞后,SAL-2和h LYZ基因的mR NA均有表达,Western blot分别检测到28.6 ku和14.7 ku目的蛋白条带。扩增纯化后经TCID50方法检测病毒滴度分别为108.17TCID50/mL、108.50TCID50/mL、108.63TCID50/mL和107.60TCID50/mL。表明成功构建包装的重组腺病毒对HEK293细胞具有较强的感染能力,可稳定介导SAL-2基因和h LYZ基因在HEK293细胞中的表达。 相似文献
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本研究选择O型FMDV VP1基因上保守序列作为可能的干扰位点,合成了siRNA,将siRNA克隆到pSIREN-Shuttle中,获得siRNA重组表达载体pShuttle-VP1。用限制性内切酶PI-Sce I和I-Ceu I双酶切siRNA重组表达载体和腺病毒质粒载体pAdeno-X,利用体外连接法获得了重组质粒。经过PCR扩增、酶切鉴定及序列测定,证明成功构建了重组腺病毒质粒pAdeno-VP1。利用脂质体转染法,将Pac I线性化的重组腺病毒质粒pAdeno-VP1转染HEK293细胞,包装成重组腺病毒rAdeno-VP1。经过PCR鉴定正确后大量增殖,增殖的重组腺病毒经过浓缩、纯化得到了高滴度的病毒。用终点稀释法测定病毒滴度为2.5×109PFU/mL。将rAdeno-VP1感染IBRS-2细胞后,12 h后再加入100TCID50的FMDV O/HKN/2003。结果显示,重组腺病毒能够抑制FMDV的复制。 相似文献
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O型口蹄疫病毒样颗粒编码基因的真核表达载体构建及表达 总被引：1，自引：0，他引：1
为了构建O型口蹄疫病毒(FMDV)病毒样颗粒编码基因的真核表达载体,将FMDV结构蛋白VP0、VP1、VP3的基因分别克隆到真核表达载体pVAX1中,再分别通过带有BsmBⅠ、EcoRⅠ和SalⅠ酶切位点的引物,分别对重组质粒pVAX-VP0、pVAX-VP1和pVAX-VP3扩增后进行酶切,用T4连接酶连接得到重组的真核表达载体pVAX-VP0VP1VP3。对重组真核表达载体pVAX-VP0VP1VP3进行PCR鉴定并测序。用脂质体将重组真核表达载体pVAX-VP0VP1VP3转染BHK-21细胞,用间接免疫荧光试验(IFA)及Western blot检测重组真核表达载体pVAX-VP0VP1VP3在细胞中的表达情况。结果表明,成功构建了重组真核表达载体pVAX-VP0VP1VP3,并在BHK-21细胞中得到表达。 相似文献
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用RT PCR 方法扩增口蹄疫病毒VP1基因,并克隆到pgem t easy 载体中,通过酶切、PCR和测序验证克隆正确,再亚克隆到真核表达载体pcDNA 3.1( )上,得到重组质粒pcDNA VP1。根据获得的口蹄疫病毒VP1基因的序列,结合相关的文献资料,设计合成免疫串联片段F,F含有VP1 基因的主要抗原位点141 位~160 位及200 位~213位的氨基酸序列,将F 片段克隆到真核表达载体pcDNA 3.1( )中,得到重组质粒pcDNA F。在脂质体介导下,重组质粒pcD NA VP1和pcDNA F转染Vero细胞。间接免疫荧光分析表明VP1 基因和F 基因均在Vero细胞中成功进行了瞬时表达,为进一步研制FMD基因疫苗奠定了基础。 相似文献
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非洲马瘟病毒VP7基因的克隆及其在昆虫细胞中的表达 总被引：1，自引：1，他引：0
为了获得具有天然活性的非洲马瘟病毒重组VP7蛋白,制备针对非洲马瘟VP7蛋白的单克隆抗体及建立快速抗体检测方法,本试验通过PCR扩增、胶回收纯化后经Xba Ⅰ和Hind Ⅲ特异性酶切,将VP7基因克隆于昆虫杆状病毒表达载体pFastBacHTB,经PCR、酶切、测序鉴定后,成功构建了携带VP7基因的重组质粒pFastBacHTB-AHSV-VP7。该重组质粒转化含有杆状病毒穿梭载体的DH10Bac感受态细胞,经抗生素、PCR筛选,获得转座的杆粒Bacmid-AHSV-VP7,并在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒,再感染Sf9昆虫细胞,收获表达产物。表达产物经Western blotting分析表明,VP7重组蛋白得到表达,其分子质量大小约为45 ku,且具有良好的生物活性。 相似文献
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应用PCR技术扩增牛新孢子虫NcSRS2基因,纯化PCR产物后与克隆载体pMD18-T Simple Vector连接,将PCR、酶切鉴定及测序分析正确的pMD-18T-NcSRS2重组质粒进行EcoRⅠ和XbaⅠ双酶切,克隆至相同酶切回收后的腺病毒穿梭载体pCR259中,再将PCR、酶切鉴定正确的pCR259-NcSRS2重组质粒转染293细胞,应用IF-AT和Western-blotting技术检测重组质粒在293细胞中的表达情况。结果显示,扩增的牛新孢子虫NcSRS2基因长度为1 227bp,与GenBank中发表的NcSRS2（AF061249）核苷酸序列同源性为99%,构建的pCR259-NcSRS2重组质粒在293细胞中得到瞬时表达,表达蛋白的相对分子质量为43 000,具有较好的反应原性。本试验为新孢子虫病腺病毒载体疫苗的构建奠定了基础。 相似文献
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《中国兽医学报》2016,(9):1476-1483
参考GenBank中登录的猪细小病毒(PPV)(M38367.1)基因序列,设计1对可以扩增出约1.6kb PPV VP2基因片段的特异性引物,上、下游引物的5′端均引入BamHⅠ酶切位点,PCR扩增得到VP2目的片段后,连接到pMD18-T载体中得到重组质粒pMD-VP2。取本实验室构建的含绿色荧光蛋白标记基因的伪狂犬病毒(PRV)通用转移质粒pG和构建好的pMD-VP2质粒,用BamHⅠ对质粒pG和pMD-VP2进行酶切,然后用CIAP对酶切产物进行去磷酸化处理,纯化回收后将VP2基因插入pG质粒中获得重组伪狂犬病毒转移质粒pGVP2。用脂质体转染法将PRV HB-98株全基因组与质粒pGVP2共转染ST细胞,得到携带有PPV VP2外源抗原基因和绿色荧光蛋白标记基因的重组伪狂犬病毒rPRV-VP2株。结合VP2基因PCR扩增和绿色荧光观察,经5轮病毒空斑纯化得到了纯化的重组病毒rPRV-VP2株。 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