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应用重叠扩增PCR(SOE PCR)技术将鸡白细胞介素15(ChIL-15)的冗长信号肽序列替换为较短的鸡白细胞介素2(ChIL-2)信号肽序列,构建了一种新型ChIL-15融合基因(NChIL-15).将其连接到克隆载体pMD18-T中得到重组克隆质粒pMD18T-NChIL-15.阳性克隆质粒经鉴定正确后将NChIL-15基因亚克隆入真核表达载体pcDNA3.1(+)中,得到重组阳性表达质粒pcDNA3.1(+)-NChIL- 15.阳性表达质粒经鉴定正确后应用磷酸钙法体外转染293T细胞,通过间接免疫荧光试验(IFA)检测到了NChIL-15蛋白在293T细胞中的表达.本研究为NChIL-15佐剂作用的深入研究奠定了基础. 相似文献
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鸡IL-18基因重组真核表达载体的构建及其表达产物的生物学活性 总被引：5，自引：0，他引：5
从pMDChIL-18克隆质粒扩增获得了ChIL-18全基因片段，并将其重组到真核表达载体pcDNA3．1( )。经酶切、质粒PCR鉴定和基因测序，鸡IL-18全基因被正确重组到pcDNA3．1( )真核表达质粒上；将重组真核表达质粒pcDNA3．1( )-ChIL-18转染COS-7细胞，转染细胞中含ChIL-18基因的mRNA。SDS-PAGE分析表明，表达产物是与ChIL-18相符的约23ku的蛋白条带。鸡淋巴细胞转化试验表明，表达产物对鸡淋巴细胞具有明显诱导转化作用。 相似文献
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本试验旨在构建猪生长激素促分泌素受体（pGHS-R）真核表达系统,并瞬时转染人源胚胎肾细胞（HEK293T）观察其表达情况。以猪基因组为模板,通过剪接重叠延伸聚合酶链式反应（SOE-PCR）克隆出pGHS-R的编码区序列,插入真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pcDNA3.1(+)/pGHS-R,酶切鉴定并测序,加myc标签,瞬时转染HEK293T细胞,用Western blotting鉴定该重组质粒是否能在真核细胞中表达相应的目的蛋白。结果显示,本试验成功扩增出pGHS-R编码序列,酶切和测序结果表明pcDNA3.1(+)-myc/pGHS-R构建正确,Western blotting方法证实转染的该质粒能在HEK293T细胞中正确表达目的蛋白。结果表明,本试验成功构建了pGHS-R真核表达载体,并正确表达蛋白,为进一步研究GHS-R的功能奠定了基础。 相似文献
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为构建鸡高迁移率族蛋白B1(Chicken high mobility group B1 protein,chHMGB1)真核表达载体,并在真核细胞中进行暂态表达和鉴定。研究酶切回收含有鸡高迁移率族蛋白全长基因的PGEM-chHMGB1质粒,将chHMGB1全长与pcDNA3.1(+)载体连接构建pcDNA-chHMGB1全长重组表达质粒。将其质粒采用脂质体转染293T细胞,进行暂态表达,利用Western blotting和间接免疫荧光法对表达的蛋白进行鉴定。结果显示,pcDNA-chHMGB1重组表达质粒克隆片段大小正确,瞬时转染293T细胞,Western blotting在细胞浆和培养上清中同时检测到相对分子量约为30 ku的目的条带;利用chHMGB1特异性抗体进行IFA试验可以检测到目的蛋白的表达。本研究成功构建了鸡高迁移率族蛋白B1的真核表达载体pcDNA-chHMGB1,并且在真核细胞中得以有效表达,从而为进一步研究chHMGB1蛋白的生物学功能提供生物材料。 相似文献
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猪白细胞介素18全基因的克隆、真核表达质粒的构建及其在猪肾细胞中的表达 总被引：2，自引：1，他引：1
通过RT-PCR方法自猪脾脏淋巴细胞中扩增mpIL-18基因.序列测定表明,pIL-18全基因核苷酸长度为579bp,编码192个氨基酸.将其克隆到真核表达载体pcDNA3.1中,构建重组质粒pcDNA-IL18m,所获重组质粒经过酶切、测序鉴定,证实含有目的片段,且连接、构建正确.阳性克隆鉴定后,在脂质体作用下转染猪肾细胞(PK15),通过提取RNA检测到了mpIL-18在PK15细胞中的表达. 相似文献
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《黑龙江畜牧兽医》2014,(15)
为了构建鸡α干扰素基因真核表达载体,试验采用RT-PCR方法从鸡胚成纤维细胞中扩增α干扰素基因序列,将扩增产物定向插入到pMD18-T克隆载体中,进行序列测定;测序正确的质粒经EcoRⅠ和SacⅠ双酶切,将目的基因片段定向克隆到真核表达载体pCAGGS中,构建重组质粒pCAGGS-IFN-α;将重组质粒转染293T细胞,使用间接免疫荧光、Western-blot等方法检测目的蛋白。结果表明:已成功扩增出582 bp大小的鸡α干扰素基因片段,测序结果与GenBank报道的序列基本一致。说明阳性重组质粒pCAGGS-IFN-α真核表达载体构建成功,能正确表达鸡α干扰素。 相似文献
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试验利用Trizol法从鸡肠道组织提取总RNA,采用特异性引物通过RT-PCR扩增出α-2,3-唾液酸转移酶Ⅰ(α-2,3-sialyltransferaseⅠ,ST3GALⅠ)基因的cDNA片段,并将其克隆至pGEM-T easy载体,获得阳性重组质粒,再以阳性重组质粒为模板亚克隆ST3GALⅠ基因的完整ORF区,定向插入到真核表达载体pcDNA3.1(+)上,进行PCR、限制性酶切和DNA序列分析鉴定。结果表明,ST3GALⅠ基因全长1029 bp,测序结果同GenBank数据库收录的序列一致,无任何密码子缺失与突变,插入到真核表达载体pcDNA3.1(+)上的目的基因大小方向均正确。本研究成功构建了pcDNA3.1(+)-ST3GALⅠ真核表达载体,为下一步的真核表达及对ST3GALⅠ基因的功能研究奠定了基础。 相似文献
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为开发可以体外合成牛促卵泡素(FSH)的表达系统,试验通过基因合成的手段获得牛FSHβ基因的目的片段,并通过酶切及T4 DNA连接酶的作用将FSHβ目的片段构建成pcDNA3.1-EGFP-FSHβ重组质粒,并针对构建的pcDNA3.1-EGFP-FSHβ重组质粒,采用菌液PCR、双酶切的方法对该重组质粒进行鉴定,并进一步利用293T细胞检测该重组质粒的表达情况。结果表明:菌液PCR和双酶切的结果证实了牛FSHβ基因片段插入了重组质粒中;pcDNA3.1-EGFP-FSHβ重组质粒可以在293T细胞中表达出绿色荧光蛋白,而且经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测发现pcDNA3.1-EGFP-FSHβ重组质粒可以在293T细胞中转录并翻译出牛FSHβ基因的蛋白表达产物。说明pcDNA3.1-EGFP-FSHβ重组表达质粒构建成功,并且可以在293T细胞中表达。 相似文献
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《动物医学进展》2021,42(7)
为了构建布鲁氏菌16M BtpA和BtpB真核表达载体,便于深入研究其在布鲁氏菌逃逸宿主免疫机制中发挥的作用。根据GenBank公布的布鲁氏菌16M BtpA和BtpB序列设计了特异性引物,并提取布鲁氏菌16M总RNA,将其逆转录成cDNA为模板,进行了PCR扩增,获得BtpA和BtpB目的片段,连接至pMD18-T进行克隆纯化,再与pcDNA3.1载体进行连接,经PCR、酶切和测序分析等方法鉴定后,利用Lipofectamine~(TM)2000脂质体转染至293T细胞,Western blot检测BtpA和BtpB蛋白的表达。结果显示,PCR扩增出了873 bp的BtpA基因片段和924 bp的BtpB基因片段,依次连至克隆载体pMD18-T和真核表达载体pcDNA3.1,经限制酶EcoRⅠ和XbaⅠ酶切验证正确,测序分析显示与GenBank公布的序列信息完全一致;Western blot检测结果显示,pcDNA3.1-BtpA-HA和pcDNA3.1-BtpB-HA分别在32 ku和35 ku处出现了条带,与预期结果完全相符,说明能在293T细胞中表达。成功构建了pcDNA3.1-BtpA-HA和pcDNA3.1-BtpB-HA真核表达载体,并证实了其能在293T细胞中表达,为后续研究其作用与机制提供了材料。 相似文献
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[目的]构建以及鉴定牛lncRNA H19过表达重组载体,为进一步探究lncRNA H19与miRNA 491和牛CART基因的互作关系奠定试验基础。[方法]NCBI获取牛lncRNA H19序列,经聚合酶链式反应(PCR)扩增,双酶切后载入pcDNA3.1-EGFP载体得到重组质粒。将pcDNA3.1-EGFP-H19、miRNA 491和CART 3种质粒共转染至HEK293T细胞,在细胞内反复扩增过表达之后,利用荧光定量技术检测pcDNA3.1-EGFP-H19的表达量。[结果]结果显示pcDNA3.1-EGFP-H19载体序列正确;293T细胞绿色荧光达50%,且强度适中,说明转染效果良好;lncRNA H19在293T细胞中显著表达。[结论]pcDNA3.1-EGFP-H19载体构建成功,试验将为后续探究lncRNA H19与miRNA 491和CART基因之间的互作关系创造试验条件。 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