青霉素G酰化酶α亚基Ser177的突变对酶活性的影响

用盒式突变和定点突变对大肠杆菌青霉素G酰化酶α亚基177位ser进行了突变研究,结果发现所挑选的突变体均无酶的活力,这一结果可能可以用来解释Ser 177附近肽段和一些青霉素结合蛋白青霉素结合区在一级结构上保持同源性的原因。     

受白粉菌诱导大麦抗感等基因系蛋白质变化的双向电泳分析

分别对接种与否的大麦抗—感白粉病等基因系—叶期幼苗取材进行蛋白质双向电泳分析。结果表明,病原的侵入使抗—感两系在30Kd以下的低分子量区域的蛋白质发生了明显变化。接种48小时之后,抗病系在pH5.5、6.0、6.8及8.8附近出现了对照中所没有的蛋白质,而在pH6.0和8.8附近的蛋白质则较对照有减小的趋势;感病系在pH6.0附近蛋白质明显增多,在pH8.8处不仅在量上有大幅度提高,而且种类也有增加。结果还表明,抗—感系间在未接种的情况下双向电泳图谱也有差异,接种之后由于感病系在pH8.8处蛋白质的特异性合成,使抗—感两系间的差异缩小。     

大麦条纹花叶病毒新疆株(BSMV-XJ)RNA_2的cDNA克隆及5′端序列分析

分离了大麦条纹花叶病毒(BSMV)新疆株基因组的3个RNA组份。以RNA2为模板,3′端互补寡核苷酸为引物,合成了第一条cDNA链和第二条cDNA链,将ds-cDNA重组在pUC9质粒中,转化大肠杆菌细胞,获得含RNA3′端的克隆,并证明所选克隆的cDNA含有新疆株几近全长的RNA2组份。对于插入片段为3.3kbp的112号克隆进行了酶谱分析,得到了与国外典型株类似的结果;用双脱氧终止法分析了相当于RNA 2 5′端250bp的cDNA酶切片段,表明与国外典型株有十分相似的一级结构。     

大肠杆菌棉子糖操纵子a-半乳糖苷酶表达的调节控制

大肠杆菌棉子糖操纵子(raf)位于质粒,其第一结构基因rafA编码的a-半乳糖苷酶为诱导酶。Rat操纵子比乳糖操纵子(lac)或蜜二糖操纵子(mel)对诱导物有更严格的结构特异性。该酶被蜜二糖或棉子糖诱导,也被D-半乳糖微弱诱导,但不受乳糖、PNPG等结构相近糖所诱导。A-半乳糖苷酶的酶诱导形成能力在对数生长末期出现高峰。Rat 操纵子基因结构组成及调节与乳糖操纵子相似。以阻遏物为中介的负调控在raf操纵子调节中起主要作用,同时以环腺苷一代谢降解物基因激活蛋白(cAMP-CAP)为中介的正调控也参与调节。当0．4％葡萄糖加入到其它碳源培养基时,该酶表达水平下降至原活力的1／2—1／3。无论诱导或组成型酶的葡萄糖抑制均未见瞬时抑制。腺苷环化酶(cya)缺失或环腺苷受体蛋白(crP)和cya双缺陷菌株的酶表达则分别下降到原活力的9％和2．5％。Cya突变株或葡萄糖对raf操纵子表达的抑制可被cAMP解除,但cya和crP双缺陷菌株仍有葡萄糖抑制,而且这种抑制不为cAMP抵消,表明通过降低cAMp而影响cAMP-CAP复合体形成还不能解释代谢降解物抑制的全部机制。尚无证据说明吲哚类小分子化合物和低浓度尿素对raf操纵子表达的明显作用。     

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)