鹦鹉热嗜衣原体分型的研究进展

鹦鹉热嗜衣原体广泛分布于世界各地,能感染禽鸟、哺乳动物及人类,严重威胁人类健康,并对世界各国的禽类养殖业造成巨大影响。传统的血清学分型法由于其特异性低、敏感性差等缺点几乎已被淘汰,随着分子生物学技术的发展,基因分型法凭借其高特异性、高敏感性、高分辨率等优点,有效地弥补了血清学分型的不足。本文就Cps分型方法的研究进展进行了综述。     

鹦鹉热嗜衣原体检测和诊断方法研究进展

鹦鹉热嗜衣原体(Cps)是一种常见的鸟类、禽类、家畜及人类病原体,快速、准确的确定病原体是控制疾病的关键。本文综述了Cps检测和诊断方法的研究进展,这些方法包括抗原检测、抗体检测和核酸检测等。     

鹦鹉热衣原体B细胞表位的表达及抗原鉴定

利用噬菌体PⅢ蛋白为载体对鹦鹉热衣原体单抗17筛选得到的B细胞抗原表位进行了研究,利用改构后的原核表达载体pQE30,构建含有B细胞表位基因的重组表达质粒,表达的蛋白经ELISA及Western Blot实验证实能够与单抗C17发生特异反应,蛋白免疫动物后得到了抗鹦鹉热衣原体的抗体,验证了所筛选表位的真实性。     

重组鹦鹉热衣原体主要外膜蛋白的抗原性研究

构建了鹦鹉热衣原体主要外膜蛋白的原核表达载体,并对其进行了诱导表达,经过纯化,复性,获得了重组蛋白。用Westen-blotting和胶体金方法检测,此蛋白具有衣原体的免疫原性。经兔免疫接种实验,获得了多抗血清,用ELISA方法检测,其抗体滴度为1：2000。     

鹦鹉热衣原体主要外膜蛋白基因序列的扩增、克隆和原核表达

主要外膜蛋白在鹦鹉热衣原体感染过程中起主要作用。扩增了主要外膜蛋白基因,克隆入pGEM-T和pET32a( ),经PCR筛选和酶切鉴定,进行诱导表达和重组蛋白的纯化与复性研究,为进一步进行鹦鹉热衣原体的诊断试剂和疫苗研究创造了条件。     

鹦鹉热衣原体重组主要外膜蛋白诱导小鼠免疫应答的观察

用鹦鹉热衣原体 (Chlamydiapsittaci,Cps)重组主要外膜蛋白 (RecombinantMajorOuter Membraneprotein ,r MOMP)免疫小鼠 ,观察小鼠免疫后对r MOMP和Cps菌体蛋白的免疫应答。r MOMP皮下注射免疫BALB/c小鼠 ,对照组仅注射佐剂。免疫前和第 3次免疫后 10d收集血清 ,以常规ELISA法检测抗体效价 ;MTT方法检测脾淋巴细胞对r MOMP和Cps菌体蛋白的特异性增殖反应。免疫组小鼠在免疫 38d后免疫血清中抗r MOMP的抗体效价可达 1∶2× 10 4,抗Cps菌体蛋白的抗体效价为 1∶4× 10 3 ;脾淋巴细胞对r MOMP和Cps菌体蛋白的增殖指数明显高于佐剂对照组 (p <0 .0 1,具有显著性意义。r MOMP在小鼠体内可诱导Cps特异性的体液免疫和细胞免疫应答 ,说明具有较好的免疫原性     

三种方法对禽鹦鹉热嗜性衣原体疑似病例病原的流行调查

目的利用免疫荧光抗体法、PCE-ELISA、PCR法分别检测北京市及周边地区禽鹦鹉热嗜性衣原体疑似病例的病原,以了解和评价该病原流行状况。方法收集临床疑似禽衣原体的父母代、商品代家禽、SPF鸡喉头拭子、气囊组织、子宫黏膜,组织标本固定后采用直接免疫荧光染色测定衣原体包含体;喉头拭子、子宫黏膜处理后以PCE-ELISA定量测定衣原体;以衣原体保守性高的序列设计CTU/CTL引物扩增病料组织的omp-1基因片段。结果直接荧光染色法显示家禽衣原体平均阳性率为38.7%,组织检出率依次为子宫黏膜、气囊、喉头拭子;PCE-ELISA检测显示平均阳性率为58.7%,其中SPF鸡阳性率为10.0%,健康肉鸡达到30.0%;PCR法检测家禽衣原体平均阳性率为71.6%,SPF鸡为10.0%。样本的目的基因与标准禽衣原体6BC同源性超过99%。结论种禽场和商品养殖场均发现疑似病例中禽鹦鹉热嗜性衣原体感染情况较为严重。荧光抗体染色、PCE-ELISA可用于临床实践中进行快速、准确检测。     

肺炎嗜衣原体蛋白酶样活性因子免疫优势区基因 重组蛋白在早期诊断中的应用

[目的]克隆和表达肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)蛋白酶样活性因子(CPAF)免疫优势区基因,评价重组蛋白在早期感染诊断中的应用价值.[方法]挑选并克隆出Cpn CPAF免疫优势区基因,构建原核表达载体,诱导表达并纯化重组蛋白,分析其抗原特异性;间接ELISA法检测Cpn参考血清、临床血清标本中的特异性IgM抗体,以及呼吸道感染患者痰咽拭子中的Cpn抗原;检测沙眼衣原体(Chlamydia trachomatis,Ct)临床阳性血清和泌尿生殖道分泌物.[结果]高效表达和纯化出一相对分子量约51.3kDa的重组蛋白;Western blot证明其只与人抗Cpn抗血清发生特异性反应;间接ELISA法检测40份Cpn IgM参考血清,阴性和阳性结果的一致率均为100%(40/40);与"金标准"方法MIF对照,检测300例临床血清标本中的IgM抗体,符合率为98.3%;与PCR试剂对照,检测120份呼吸道感染患者痰咽拭子中的Cpn抗原,符合率为88.3%;检测Ct阳性血清和泌尿生殖道分泌物,与Ct没有交叉反应.[结论]制备的CPAF免疫优势区基因重组蛋白具有良好的抗原性,在Cpn感染早期诊断中具有较高的利用价值.     

鹦鹉热衣原体MIP蛋白生物信息学分析

为初步了解鹦鹉热衣原体(Chlamydia psittaci,Cps)巨噬细胞感染增强蛋白(macrophage infection po-tentiator,MIP)的生物学特征.本研究从美国国家生物技术信息中心(national center for biotechnology infor mation,NCBI)...     

肺炎嗜衣原体主要外膜蛋白的克隆表达与抗原性研究

肺炎嗜衣原体主要外膜蛋白是其特征抗原之一。实验中通过PCR方法从肺炎嗜衣原体基因组中扩增主要外膜蛋白基因,插入pET32a(+)表达载体,转化BL21(DE3)感受态细胞,得到表达56kD融合蛋白的工程菌株。该菌株的表达量可达53%,提纯后的主要外膜蛋白纯度可达90%以上,在Western Blotting试验和胶体金免疫层析试验中显示了良好的抗原性。     

Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major

Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDK) are involved in the salvage pathway by which free purines are converted to nucleosides and subsequently to nucleotides. In this report, we describe the cloning of NDK coding-sequence from Leishmania major, the expression of the enzyme containing a His(6)-tag in Escherichia coli, and purification of the catalytically active native protein by affinity chromatography using Ni-NTA resin.     

Expression and purification of the recombinant His-tagged GST-CD38 fusion protein using the baculovirus/insect cell expression system

CD38 is a type II transmembrane glycoprotein found in myriad mammalian tissues and cell types. It is known for its involvement in the metabolism of cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. CD38 itself has been shown to have clinical significance in certain diseases with possible utilization in diagnostic and prognostic applications. Previous studies on several autoimmune diseases have shown the usefulness of recombinant CD38 protein expressed from Escherichia coli and Pichia pastoris in the detection of autoantibodies to CD38 via Western blot and ELISA. In this study, we produced a 6 x His-tagged GST-CD38 fusion protein using a recombinant baculovirus/insect cell expression technique that was purified as a soluble protein. The fusion protein was purified to homogeneity by affinity and gel filtration chromatography steps. It has an apparent molecular mass of 56 kDa on SDS-PAGE gel stained with Coomassie blue and was recognized on Western blots by antibodies against human CD38 as well as the polyhistidine tag. Peptide mass fingerprinting analysis confirmed the identity of human CD38 in the fusion protein.     

Expression, purification, characterization of human 3-methylcrotonyl-CoA carboxylase (MCCC)

The current study reports the use of baculovirus system to express functionally active human recombinant 3-methylcrotonyl-CoA carboxylase (MCCC), a heteromultimeric complex that is composed of alpha and beta subunits which are encoded by distinct genes. Using immuno-affinity purification, an efficient protocol has been developed to purify the active MCCC which appears to reside in a approximately 500-800kDa complex in Superpose-6 gel-filtration chromatography. Consistent with the native enzyme, in the recombinant human MCCC, the stoichiometry of alpha and beta subunits are at a one:one ratio. The k(cat) value of the recombinant enzyme is determined to be approximately 4.0s(-1). It also possesses K(m) values (ATP: 45+/-11microM; 3-methylcrotonyl-CoA: 74+/-7microM) similar to those reported for the native enzyme. The recombinant human MCCC described here may provide a counter-screen enzyme source for testing cross reactivity for inhibitors against acetyl-CoA carboxylases which are designed to treat obesity, type 2 diabetes and other metabolic disorders.     

人组蛋白α-氨基乙酰基转移酶Nat11表达纯化、晶体生长及底物结合研究

α-氨基乙酰基转移酶11(Nat11)催化组蛋白H4和H2A氨基端乙酰化修饰,发挥着重要的表观遗传调控功能。将人Nat11基因构建到原核表达载体p SUMO中,转化入大肠杆菌BL21(DE3)进行重组表达。通过镍柱亲和层析等一系列体外纯化步骤,获得高纯度Nat11。利用等温滴定量热法(ITC),测得Nat11与底物多肽微摩尔量级结合常数。利用质谱技术,发现纯化后的Nat11结合有大肠杆菌内源产生的乙酰辅酶A或辅酶A,在ITC滴定过程中可以产生对多肽底物的乙酰化修饰,表明纯化获得的Nat11在溶液中具有酶活力。随后,对Nat11进行晶体生长研究,通过初筛优化获得蛋白截短体及底物-酶融合蛋白单晶。     

影响农杆菌渗入法瞬间表达重组蛋白系统的若干因素

利用农杆菌渗入法(Agroinfiltration)在植物中瞬间表达重组蛋白的系统,具有高效、安全、简便的特点,是一种重要的生产重组蛋白的方法。概述了Agroinfiltration系统的建立、发展及在生产重组蛋白领域的应用进展;并对影响该系统的技术因素作了深入探讨。     

Expression, purification and anti-tumor activity of curcin

Curcin,purified from the seeds of Jatropha curcas,can be used as a cell-killing agent.Understanding the anti-tumor activity of the recombinant protein of curcin is important for its application inclinical medicine.The segment encoding the mature protein of curcin was inserted into Escherichia colistrain M 15,and the recombinant strain was induced to express by isopropyl-β-D-thiogalactopyranoside at aconcentration of 0.5 mM.The recombinant protein was expressed in the form of inclusion bodies andpurified by Ni-NTA affinity chromatography.The target protein was incubated with the tumor cells atdifferent concentrations for different times and the results demonstrated that the target protein could inhibitthe growth of tumor cells (NCL-H446,SGC-7901 and S180) at 5μg/ml.     

重组蛋白在中国仓鼠卵巢细胞中高效表达的影响因素

高效表达重组蛋白 ,对于生物制药意义重大。大多数药用蛋白是糖蛋白 ,中国仓鼠卵巢细胞 (Chinesehamsterovarycell,CHO)是目前重组糖基蛋白生产的首选体系。影响外源蛋白在CHO细胞中表达的因素很多 ,从CHO细胞表达体系、表达载体系统、外源基因、表达细胞株的加压扩增与筛选、细胞大规模培养等方面对CHO高效表达加以阐述 ,同时提出存在的问题和未来的发展方向。