乙型肝炎病毒表面抗原基因在杆状病毒载体与昆虫细胞体系中的表达

根据杆状病毒A组代表种苜蓿Y纹夜蛾核多角体病毒(Autographa californica Nuclear Po-lyhedrosis Virus,简称AcNPV)的分子生物学资料,我们采用分子剪裁和拼接手段,对AcNPV多角体蛋白基因加以修饰,并用人工合成定位探针,获得大约在该基因的ATG转译起始密码子一9处加有合成BamHl连接序列的转移载体质粒pAc—MV。 再与从乙肝病毒adw亚型的表面抗原基因HBng亚克隆株pYPss一1分出带有Ban·Hl粘性末端的HBsAg基因,构建表达载体质粒pAc—MV—HBsAgo经与野生型AcNPV DNA对Spodoptem 7rugJPerda细胞共转染,借助同源交换,获得插有HBsAg基因的多角体缺陷的重组病毒。根据HBsAg诊断血球凝集试验和HBs^g诊断酶标免疫测定,表明重组病毒使HBsAg基因在昆虫细胞中得到了表达,免疫电镜显示表达产物呈球状颗粒,大小约为22nm。表达产物粗制品按1μg／鼠对bal b／c鼠免疫,并于三周后强化免疫能产生抗体。由HBsAg纯样品酶标免疫法标定的标准曲线估计每升培养物的表达产物约{一8mg,细胞量为1一2×106个/ml.用重组病毒感染玉米螟4龄幼虫,也获得HBsAg基因的表达,展示了简易生产HB sag诊断试剂和疫苗的可喜前景。     

毒素源性大肠杆菌K88ac抗原基因的次级克隆和重组质粒的限制图

从我国引起仔猪腹泻的野生株E. coli 79-1454克隆了K88ac抗原基因,获得了重组体E. coli RR1(pNZ8801),分子量为10 Md。为了得到分子量更小的重组体,用E．coRI消化重组质粒,除去中间约3.2 Md的片段,得到次级克隆株E. coli RR1(pNZ8802),质粒分子量为6.8 Md。测定其K 88ac抗原为阳性,而且其产生的K88at抗原量略高于初级克隆株。电镜照片表明重组体表面长有菌毛。萤光抗体染色和猪刷状缘细胞粘附试验亦表明有良好的粘附生物活性。此重组体可以与本人克隆的K99抗原基因构建成复合重组体。本文还作了重组体的限制性酶切图。     

大肠杆菌棉子糖操纵子α—半乳糖苷酶表达的调节控制

The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS)     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

酵母ENO2上游激活顺序对酵母 LEU2表达的影响

本文将酿酒酵母(Saccharomyces cerevisiae)中编码烯醇化酶的基因之—ENO2的上游墩活顺序嵌入穿梭质粒YEpl3上酵母LEU2基因上游—405Hpa I的酶切部位。LEU2为编码β-异丙基苹果酸脱氢酶基因。从而研究了酵母ENO2的上游激活顺序对酵母LEU2表达的影响。实验结果表明ENO2上游激活顺序不论正向或反向嵌八都激活LEU2的表达达四倍左右。有亮氨酸存在的条件下,LEU2的表达受到抑制。ENO2上游激活顺序在对LEU2表达的激活上并不受葡萄糖的诱导。提出了用ENO2上游激活顺序组建高表达系统的可能性。     

小麦高分子量谷蛋白亚基基因片段的体外扩增

生物体尤其是高等动植物,绝大多数基因的拷贝数是很低的,如果没有基因扩增的手段,几乎无法研究基因的结构及其与表达的关系。当今分子生物学发展中最基本的技术之一基因克隆,实际上就是基因体内扩增(in vivo amplification)的技术。近年来发展了一种基因体外扩增(in vitro amplification)的新技术,又称     

几种固氮菌nifA基因片段的同源性分析

参照已知数种固氮菌nifA基因的DNA序列,选择其中间区域的两个保守性较强的序列合成引物,利用肺炎克匠杆菌(Klebsiella pneumoniaef)、棕色固氮菌(Azotobacter vinela-ndii)、巴西固氮螺菌(Azospirllum brasilense)、草螺菌(Herbaspirillum seropedicae)和深红红螺菌(Rhodospirillum rubrum)的总DNA进行聚合酶链反应,结果均扩增出约450bp大小的片段,经证实为各种固氮菌的nifA基因部分片段。 核酸印迹分子杂交结果显示出nifA基因在不同固氮菌中的同源性不强。     

脉冲凝胶电泳系统及其应用

脉冲凝胶电泳系统是近年来发展起来的一种分离大分子染色体DNA的新型电泳技术。本文介绍了该技术的基本原理以及各种脉冲凝胶电泳系统,并比较了各个系统的优缺点。简要介绍了该技术在酵母、寄生虫以及高等动物染色体DNA的核型分析与基因定位中的应用。