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依据猫IFN-ω3和THY-α1的分子结构特征及大肠杆菌密码子的偏爱性设计引物,同时在IFN-ω3和THY-α1中间设计了1个(Gly-Gly-Gly-Gly-Ser)×3总共15肽的linker.采用搭桥PCR方法获得二者的融合基因.将该基因克隆至pBVIL载体中,构建了pBVIL-IFNω3-THYα1原核表达载体.将重组栽体转化至Rosseta(DE3)细胞中,经过IPTG诱导表达及蛋白质分离纯化,获得了质量浓度为0.78 g/L的IFNω3-THYα1融合蛋白.经E玫瑰花环形成试验和细胞病变抑制试验,结果表明E玫瑰花环形成率平均为23.4%,融合蛋白的活力值为7.4%,细胞病变抑制试验同阳性对照组结果基本一致,融合蛋白中的IFNω3、THYα1均较好地保持各自的生物活性,为IFNω3-THYα1融合蛋白进一步的开发和应用奠定了基础. 相似文献
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通过构建鸡α干扰素真核表达载体,以获得能够表达鸡α干扰素的酵母工程菌株。首先根据GenBank公布的鸡α干扰素基因序列设计上、下游引物,以略阳乌鸡基因组DNA为模板,PCR扩增鸡α干扰素基因片段。然后,利用基因重组技术将鸡α-干扰素基因插入真核表达载体,通过菌落PCR和DNA测序确定目标载体,构建成功后转化至酿酒酵母AH109,再通过营养缺陷培养、质粒提取和PCR鉴定带JMB440-ChIFN-α载体的重组酵母菌株。最终通过酵母培养、总蛋白收集、SDS-PAGE电泳和Western blot等,确定鸡α干扰素基因能够与酵母菌株重组,并表达α干扰素,蛋白条带大小约为23ku,与预期结果一致,表明成功构建了表达鸡干扰素基因的工程酵母菌株。 相似文献
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[目的]利用大肠杆菌原核表达系统对猪干扰素α-1(Po IFNα-1)基因进行表达,制备Po IFNα-1重组蛋白多克隆抗体。[方法]根据Gen Bank发表的Po IFNα-1核苷酸序列,合成密码子优化的Po IFNα-1基因,构建Po IFNα-1基因的p ET-28a原核表达载体,转化大肠杆菌BL21(DE3)宿主菌后,利用IPTG进行诱导表达。表达的Po IFNα-1重组蛋白纯化后,免疫昆明系小鼠,制备多克隆抗体。采用Western blot技术检测Po IFNα-1重组蛋白的多克隆抗体与天然Po IFNα-1的反应性。[结果]合成的Po IFNα-1基因在大肠杆菌BL21(DE3)宿主菌中成功表达,针对Po IFNα-1重组蛋白的多克隆抗体能够识别天然的猪干扰素α-1。[结论]合成的Po IFNα-1基因在大肠杆菌中获得了成功表达,为后续研究奠定了基础。 相似文献
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为可溶性表达重组鸡粒细胞巨噬细胞集落刺激因子(ChGM-CSF)与猪干扰素α1(PoIFNα1)融合蛋白,并研究其生物学活性,本研究分别提取鸡、猪肝脏细胞总RNA,采用RT-PCR方法扩增ChGM-CSF和PoIFNα1基因,经linker连接上述两种基因后将其克隆于pET-32a原核表达载体,转化E.coliBL21(DE3)菌株进行诱导表达,SDS-PAGE和western blot检测融合蛋白表达产物。在ST细胞/VSV病毒测定系统以细胞病变抑制法滴定该融合蛋白的抗病毒活性;同时用MTT法检测其对鸡淋巴细胞增殖活性的促进作用。结果显示,PCR扩增并融合后的ChGM-CSF和PoIFNα1融合基因约为1000bp,构建重组表达载体后,诱导表达的rChGM-CSF-PoIFNα1融合蛋白分子量约55ku,主要存在于破碎菌体的上清中,表达量较高。Westernblot检测结果显示,该融合蛋白分别能够与ChGM-CSF多抗和PoIFNα单克隆抗体特异性结合,其抗病毒比活性约为1.1×10^6IU/mg,并且具有促进鸡淋巴细胞增殖的活性。本研究为rChGM-CSF-PoIFNα1重组融合蛋白的研制及其相关活性的测定提供实验依据。 相似文献
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《中国兽医杂志》2016,(8)
根据大肠杆菌密码子的偏好性对Gen Bank中发表的鸡α干扰素基因序列进行了密码子优化,全基因合成了鸡α干扰素基因片段486 bp。构建原核表达质粒p ET-23b-Ch IFN-α。将重组质粒转化大肠杆菌BL21(DE3),用IPTG进行诱导表达,表达产物主要以包涵体形式存在,包涵体进行变性、复性和镍柱亲和纯化。表达产物经SDS-PAGE、WesternBlot分析表明,Ch IFN-α蛋白得到了高效表达,其蛋白分子质量约19 k D,经镍柱亲和纯化后获得了高纯度的重组鸡α干扰素蛋白,其含量为0.82 mg/m L。本研究为鸡α干扰素的生物学活性分析和临床产品研发奠定了基础。 相似文献
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为构建表达胸腺肽和鸡γ干扰素融合蛋白(Tα1-cIFN-γ)的重组乳酸乳球菌并检测其作为口服免疫增强剂对鸡外周血单个核细胞(PBMC)的增殖活性及鸡肠道菌群的影响,本研究将鸡胸腺肽(Tα1)和鸡干扰素(cIFN-γ)的编码基因序列(去除干扰素信号肽编码基因序列),并按照宿主菌乳酸乳球菌NZ3900株的基因组优化密码子后,以经G4S-linker连接的Tα1-cIFN-γ基因为模板,采用PCR扩增携带表达载体p NZ8149同源臂及HA标签的目的基因序列(Tα1-cIFN-γ-HA);通过PCR扩增携带Tα1-cIFN-γ-HA基因序列同源臂的全长p NZ8149载体序列,利用同源重组的方式将这两段基因连接构建重组质粒p NZ8149-Tα1-cIFN-γ-HA,并经PCR和测序鉴定正确后电转化乳酸乳球菌NZ3900感受态细胞中,构建重组乳酸乳球菌p NZ8149-Tα1-cIFN-γ-HA/NZ3900 (r L.lactis-Tα1-cIFN-γ)并经PCR和测序鉴定;将重组乳酸乳球菌r L.lactis-Tα1-cIFN-γ经乳酸链球菌素(Nisin)诱导6 h～7 h,离心超声破... 相似文献
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牛干扰素可用于对各种类型牛传染性疾病的预防和治疗。利用家蚕杆状病毒表达系统表达牛α干扰素(BoIFN-α)及牛λ1干扰素(BoIFN-λ1)。首先对BoIFN-α和BoIFN-λ1的编码基因序列进行优化合成,优化合成的BoIFN-α和BoIFN-λ1基因的密码子适应指数(CAI)值提高,更适合在家蚕中表达,且翻译后的氨基酸序列与原始氨基酸序列完全一致。将优化合成的2个基因序列分别克隆至杆状病毒转移载体pVL1393,与缺失了orf1629的亲本杆状病毒BmBacmid共转染Bm N细胞,通过同源重组将BoIFN-α和BoIFN-λ1基因插入到杆状病毒多角体蛋白基因启动子下游,获得重组病毒rBmBac-BoIFN-α和rBmBac-BoIFN-λ1。用纯化的重组病毒感染家蚕5龄幼虫融合表达BoIFN-α和BoIFN-λ1干扰素,并通过PCR鉴定了2个目标蛋白基因在蚕体内的复制。采用微量细胞病变抑制法在Vero/VSV*GFP表达系统检测蚕体中表达重组BoIFN-α的效价可达1.0×10~6U/mL左右,重组BoIFN-λ1的效价可达5.01×10~4U/m L左右。试验结果提示,利用家蚕杆状病毒表达系统在蚕体内表达牛α干扰素及牛λ1干扰素是生产牛干扰素制剂更加廉价而高效的途径。 相似文献
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鸡α干扰素基因的原核表达及其活性测定 总被引：1，自引：0，他引：1
应用RT-PCR技术,从被刺激诱导的鸡脾脏淋巴细胞中扩增鸡α-干扰素(ChIFN-α)基因cDNA,经克隆和测序后,构建原核表达重组质粒pGEX-proChIFN-α(全长序列)和pET-ChIFN-α(不含信号肽序列),并分别转化相应的表达菌.重组菌经IPTG诱导,SDS-PAGE结果表明,ChIFN-α基因均获得表达,重组蛋白大小分别为45 ku、26ku左右,表达的蛋白以包涵体形式存在.表达产物经变性、提纯、复性,重组蛋白的含量约为1 mg/mL.复性后的ChIFN-α能够在鸡胚成纤维细胞上抑制H5N1禽流感病毒的复制;用水泡性口炎病毒检测结果表明,重组ChIFN-α具有较高的抗病毒作用,其生物学活性达到2×106 u/mg. 相似文献
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根据Genbank中发表鸡α-干扰素基因序列设计一对引物,采用PCR法从SPF鸡的全血白细胞中克隆了α-干扰素基因(ChIFN-α)。序列分析结果显示,与已发表的ChIFN-α的同源性在98.1%以上,与其他禽类的IFN-α同源性在67.4%以上,其中与火鸡IFN-α同源性在89.8%,而与鸭α干扰素同源性仅为67.4%。将该基因连接到原核表达载体pET-28a上,构建重组表达质粒,转入BL21,IPTG诱导后SDS-PAGE分析,可见一条约19ku的清晰蛋白带,结果表明克隆的ChIFN-α基因在原核中获得表达。 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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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