牛泡沫病毒（BSV）3026毒株的分离及分子生物学鉴定

从一头牛免疫缺陷病毒（ＢＩＶ）检测阳性３０２６号牛外周血中,分离到一株病毒,即３０２６病毒株。体外细胞增减和反转录分析证明,此病毒是一反转录病毒。可胎牛肺细胞中引起典型的泡沫样病变,形成合胞体,ＰＣＲ扩增和Ｓｏｕｔｈｅｍ杂交显示,此病毒的ＣＤＮＡ和ＰＣＲ产的均可与牛泡沫病毒（ＢＳＶ）阳性对照的ＨｉｒｔＤＮＡ杂交。３‘ＬＴＲ上游一段３３０ｂｐ的ＰＣＲ产物序理分析表明,３０２６毒株与ＢＳＶ阳性对照相比     

牛泡沫病毒反式激活因子在内部启动子上应答元件的研究

泡沫病毒的基因转录依赖至少两个不同的启动子：ＬＴＲ调节病毒结构蛋白的表达,而内部启动子（ＩＰ）则起始调节蛋白ｍＲＮＡ的转录。牛泡沫病毒（ＢＦＶ）在ｅｎｖ与３’ＬＴＲ之间有两个重叠的开放阅读框架ｏｒｆ－１和ｏｒｆ－２,分别编码ＢＦＶ ＯＲＦ－１、ＯＲＦ－２等多种调节蛋白。这此蛋白中ＢＦＶ ＯＲＦ－１为转录激活因子,称为Ｔａｓｏ Ｔａｓ对ＬＴＲ及ＩＰ均有反式激活作用。ＢＦＶ中第二类启动子ＩＰ的存在反映     

牛泡沫病毒调节蛋白功能及其在LTR上应答元件的研究

牛泡沫病毒（ＢＦＶ）具有复杂的基因组结构,其基因组除编码反转录病毒共有的ｇａｇ、ｐｏｌ、ｅｎｖ三个结构基因之外,在其ｅｎｖ和３’ＬＴＲ之间有两个重叠的读码框（ＯＲＦ－１和ＯＲＦ－２）,编码Ｂｏｒｆ－１、Ｂｏｒｆ－２、Ｂｅｔ等多种调节蛋白,其中Ｂｏｒｆ－１（２４９ａａ）为ＢＦＶ反式激活因子（Ｔａｓ）。为研究Ｂｏｒｆ－１的结构与功能,Ｂｏｒｆ－１在ＬＴＲ上的应答元件及作用机制,Ｂｏｒｆ－２、Ｂｅｔ等调     

牛病毒性腹泻病毒（BVDV）对牛免疫缺陷病毒（BIV）的激活作用

通过合胞体分析和反转录酶活力测定,首次证明牛病毒性腹泻病毒能激活牛免疫缺陷病毒的复制与表达,并进一步通过转染实验和凝胶电泳漂移分析证明,当ＢＩＶ ＬＴＲ的ＮＦ－ｋ Ｂ区缺失时,ＢＶＤＶ则不能实现其激活作用,ＢＶＤＶ直接或间接诱导牛ＮＦ－ｋＢ因子作用于ＢＩＶ ＬＴＲ的ＮＦ－ｋＢ区实现其激活作用。     

牛泡沫病毒内部启动子的克隆及功能分析

以该实验室分离并鉴定的牛泡沫病毒（ＢＦＶ３０２６）为材料,用ＰＣＲ方法首次克隆了位于ｅｎｖ基因３’端的内部启动了,经序列分析后,引入ｌｕｃ基因作瞬时分析。结果表明,该内部启动子不但基础活性高于ＬＴＲ,而且转录活怀在Ｔａｓ参与下被大大激活,其激活活性也远远高于ＬＴＲ。同源分析表明,非灵长类泡沫病毒内部启动子之间的同源性高于其与灵长类泡沫病毒内部妄动子之间的同源性。     

牛疱疹病毒Ⅰ型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活作用

由含有ＢＨＢＶ－１（ＢｏｖｉｎｅＨｅｒｐｅｓＶｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＢＩＣＰＯ（ＢＨＶ－１ＩｎｆｅｃｔｅｄＣｅｌｌＰｒｏｔｅｉｎＯ）的ＤＮＡ序列至表达载体ｐＳＶＫ３,构建质粒ｐＳＶ２．９。将该质粒与ｐＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶＬＴＲ启动子控制下的荧光素酶基因的表达。根据ｐＳＶ２．９与含有ＢＩＶＬＴＲ不同区段缺失的质粒ｐＤ－３１９－Ｌｕｃ、ｐＤ－１１５－Ｌｕｃ、ｐＤ－５２－Ｌｕｃ共转染小牛肺细胞的实验结果,推测ＢＩＶＬＴＲ－３１９位上游区的ＤＮＡ序列影响ＢＩＣＰＯ基因产物对ＢＩＶＬＴＲ表达的激活作用。     

牛泡沫病毒两类启动子活性的比较和机制探讨

牛泡沫病毒 (Ｂｏｖｉｎｅｆｏａｍｙｖｉｒｕｓ,ＢＦＶ)属反转录病毒科泡沫病毒属。其基因组两端为长末端重复序列 (Ｌｏｎｇｔｅｒｍｉｎａｌｒｅｐｅａｔ,ＬＴＲ) ,中间部分除ｇａｇ、ｐｏｌ、ｅｎｖ三个结构基因外 ,还有两个重叠的读码框ＯＲＦ 1、2 ,起始于ｅｎｖ基因 3′端 ,终止于 3′ＬＴＲ ,编码Ｂｏｒｆ 1、Ｂｏｒｆ 2等多种调节蛋白[1～ 3 ] 。其中Ｂｏｒｆ 1为ＢＦＶ的反式激活因子 ,可显著激活ＢＦＶＬＴＲ启动子起始的基因表达。近年来 ,在泡沫病毒家族成员人泡沫病毒 (Ｈｕｍａｎｆｏａｍｙｖｉｒｕｓ,ＨＦＶ)和猴泡沫病毒(…     

鸡传染性支气管炎病毒S1基因在昆虫细胞中表达水平初探

将含有鸡传染性支气管炎病毒 Ｓ１ 基因ｃ Ｄ Ｎ Ａ 的重组转移质粒ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３ Ｓ１ ． Ｈｏｌｔｅ 和ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３／４ Ｓ１ ． Ｈｏｌｔｅ 分别与粉纹夜蛾核型多角体病毒 Ｔｎ Ｎ Ｐ Ｖ Ｓ Ｖ Ｉ－ Ｇ Ｄ Ｎ Ａ（ Ｏ Ｃ Ｃ－ ,ｇａｌ＋ ） 共转染草地夜蛾（ Ｓｆ９） 细胞,经空斑纯化得到重组病毒 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 和 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 。将重组毒株分别感染 Ｔｎ５ Ｂ１ 细胞,并进行 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 与 Ｗｅｓｔｅｒｎｂｌｏｔ 检测。结果表明, Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 在感染的细胞中高效表达了 Ｓ１ 蛋白, Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 凝胶薄层色谱分析结果显示,感染病毒后７２ ｈ Ｓ１ 蛋白的表达量占细胞内总蛋白量的３５ ．８ ％ ,而 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 感染的细胞内检测不出 Ｓ１ 蛋白。经分析认为这一差异主要来自 Ｓ１ 基因翻译起始位点及其附近的周围环境。     

牛免疫缺陷病毒长末端重复序列(BIV一LTR)在大肠杆菌中的启动子功能

牛免疫缺陷病毒（ＢＩＶ）的长末端重复序列（ＬＴＲ）含有病毒的启动子,调控病毒在真核细胞中的表达。我们将ＢＩＶＬＴＲ与萤火虫荧光素酶基因连接构建成重组质粒ｐＢＩＶ一Ｌｕｃ,该质粒能在Ｅｃｏｌｉ中有效地表达出荧光素酶的活性,从而证明ＢＩＶＬＴＲ在大肠杆菌中也具有启动子功能。Ｍｕｎｇｂｅａｎ核酸酶作图分析发现,ＢＩＶＬＴＲ在Ｅｃｏｌｉ中的转录起始位点位于Ｕ_３区,而不是在真核细胞中的Ｕ＿３一Ｒ交界处。同时我们也证实了ＢＩＶＬＴＲ在Ｅ,ｃｏｌｉ中仍可被ＢＩＶｔａｔ蛋白特异性地反式激活,为研究ｔａｔ蛋白的作用机制提供了一条新的途径。     

牛疱疹病毒I型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活…

由含有ＢＨＶ－１（Ｂｏｖｉｎｅ Ｈｅｒｐｅｓ Ｖｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＩＣＰＯ（ＢＨＶ－１Ｉｎｆｅｃｔｅｄ Ｃｅｌｌ Ｐｒｏｔｅｉｎ Ｏ）的ＤＮＡ序列至表达载体ｐＳＶＤ３,构建质粒ｐＳＶ２．９。将该质粒与ＰＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶ ＬＴＲ启动子控制下的荧光素酶基因的表达。根据ＰＳＶ２．９与含有Ｂ     

精胺对牛肝tRNA~(lle)氨基酰化的刺激作用

本实验用纯化的牛肝异亮氨酸tRNA(tRNA~(Ile))和异亮氨酰tRNA合成酶(IleRS),研究了精胺对Ile-tRNA复合物形成及IleRS活性的作用。结果表明:精胺能特异地促使牛肝tRNA~(Ile)氨基酰化反应;对IleRS活性无影响;能明显地增加形成Ile-tRNA复合物反应的Vmax和tRNA~(Ile)的Km值。     

A Short and Simple Improved-Primer Extension Preamplification (I-PEP) Procedure for Whole Genome Amplification (WGA) of Bovine Cells

Embryo transfer is a reproductive technique that has a major impact on the dissemination of economically important genes and the rate of genetic gain in breeding schemes. In recent years, there has been increasing interest in the use of sexed and genotyped embryos in commercial embryo transfer programs. Marker/gene assisted selection (MAS / GAS) projects can be performed in the pre-implantation stage through mass production of characterized embryos. Biopsy of a few cells in the morulla stage is essential for pre-implantation genetic diagnosis (PGD), in which sex determination, evaluation of disease genes, and genotyping for candidate genes are performed. Limited quantity of cells and low amount of DNA restrict the use of multiple molecular analyses in PGD programs. Recently, whole genome amplification (WGA) techniques promise to overcome this problem by providing sufficient input DNA for analysis. Among several techniques proposed for WGA, the primer extension pre-amplification (PEP) and the improved-primer extension pre-amplification (I-PEP) methods are the most commonly used. However, these methods are time-consuming and need more than 12 h amplification cycles. Since the time is a critical parameter in the successful characterized embryo transfer, the shortening of diagnosis time is highly desirable. In this study, we developed a short and simple I-PEP procedure (～3 h) and evaluated its performance for the amplification of bovine genomic DNA. We assessed short WGA procedure by polymerase chain reaction (PCR) amplification of 7 specific loci. The results indicated that the short procedure possesses enough sensitivity for the molecular genetic analysis of 1 input cell. Although the efficiency of the method was 100%, there was an inconsistency between genomic DNA (gDNA) and whole genome amplification product (wgaDNA) genotypes for kappa-casein locus; that is, however, most likely due to allele drop-out (ADO) or false homozigocity. The results of this study indicate that with the application of reliable methods, WGA-amplified bovine DNA will be a useful source for sexing and genotyping bovine embryos in several quantitative trait locus (QTL) markers.     

JDV与三种牛反转录病毒相互关系的初步研究

为研究JDV与其它三种牛反转录病毒BIV、BLV、BFV的相互作用关系,将以JDV、BIV、BLV、BFV的LTR为启动子,以Luc为报告基因的质粒和以上病毒反式激活因子的表达质粒共转染BLl2细胞系,通过瞬时表达分析试验证明了JDV和BIV的LTR和Tat之间亲缘关系很近,能够相互激活;JDV Tat可以反式激活BLVLTR,BLVTax不能激活JDVLTR;JDVLTR上存在BFVTas的应答元件;BLV、BFV和BIV的LTR和反式激活因子问不存在相互激活。     

BSA载体蛋白抗体的去除

为了去除抗血清中BSA载体蛋白产生的抗体,一根对BSA载体蛋白抗体高度特异性的亲和柱被构建。结果表明：所构建的亲和柱对BSA载体蛋白抗体具高度特异性和亲和力,能有效地去除BSA载体蛋白产生的抗体。     

JDV与三种牛反转录病毒相互关系的初步研究

为研究JDV与其它三种牛反转录病毒BIV、BLV、BFV的相互作用关系,将以JDV、BIV、BLV、BFV的LTR为启动子,以Luc为报告基因的质粒和以上病毒反式激活因子的表达质粒共转染BL12细胞系,通过瞬时表达分析试验证明了JDV和BIV的LTR和Tat之间亲缘关系很近,能够相互激活;JDV Tat可以反式激活BLVLTR,BLV Tax不能激活JDV LTR;JDV LTR上存在BFV Tas的应答元件;BLV、BFV和BIV的LTR和反式激活因子间不存在相互激活.     

牛免疫缺陷病毒反式激活因子功能域的研究

牛免疫缺陷病毒 (Ｂｏｖｉｎｅｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙｖｉｒｕｓ,ＢＩＶ )与人免疫缺陷病毒 (Ｈｕｍａｎｉｍ ｍｕｎｏｄｅｆｉｃｉｅｎｃｙｖｉｒｕｓ,ＨＩＶ)同属反转录病毒科慢病毒属[1] 。ＢＩＶ基因组 5′端的长末端重复序列 (ＬＴＲ)起始病毒结构基因和非结构基因的转录[2 ] ,因而许多细胞因子和病毒编码的调节蛋白作用于ＬＴＲ ,以调节ＢＩＶ的基因表达。其中Ｔａｔ蛋白是ＢＩＶ的反式激活因子 ,可大大提高ＬＴＲ的转录水平 ,在ＢＩＶ的基因表达及基因组复制的调节中起重要作用[3 ] 。ＨＩＶ、马传染性贫血病毒 (Ｅｑｕｉ…     

Serum-free produced Bovine Herpesvirus type 1 and Bovine Parainfluenza type 3 virus vaccines are efficacious and safe

The studies described in this report were performed to determine, whether it is possible to produce live virus vaccines without serum or fractions thereof used during any cell or virus passage, thus completely serum-free. Two viruses were included in the experiments: Bovine Herpesvirus 1 (BHV-1) and Bovine Parainfluenza type 3 virus (PI3). Both viruses were found to grow to satisfactory titers, and to be stable after freeze-drying and subsequent storage at temperatures of +4 °C and −20 °C for at least one year. Moreover, a vaccine containing serum free produced BHV-1 was tested in a vaccination-challenge experiment. For comparison, a vaccine batch with BHV-1 grown in serum-containing cell culture medium was included in the study. Both vaccine preparations performed equally well and both met the strict requirements as laid down in the European Phamacopeia. Moreover, in two separate experiments the safety of serum-free produced BHV-1 and PI3 after overdose and repeated administration even in very young calves and even after four administrations has been demonstrated. This report is the first, which to our knowledge demonstrates the safety and efficacy of serum-free produced live vaccines in the target animal as well as the stability of these products. This revised version was published online in September 2006 with corrections to the Cover Date.     

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the B...