Synthesis and Expression in Escherichia coli of a Human Neutrophil Activating Protein-1/Interleukin-8 Gene

The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was sho     

丝氨酸蛋白酶抑制剂超家族的一个新成员——痘苗病毒(天坛株)基因组HindⅢK片段编码的K1蛋白

本文测定了位于痘苗病毒(天坛株)基因组的Hind Ⅲ K片段左端,由1893个碱基所组成的开放读码框架K1(ORF K1)的核苷酸排列顺序,并利用微机对ORFK1所编码K1蛋白的序列进行了研究。结果发现,K1蛋白与丝氨酸蛋白酶抑制剂超家族的成员在蛋白质一级结构上有着显著的同源性,通过点阵分析和序列同源排列,证明K1蛋白是丝氨酸蛋白酶抑制剂超家族的一个新成员,它可能具有与丝氨酸蛋白酶抑制剂超家族其他成员相似的活性中心及其他特征序列。这一发现对于研究痘苗病毒的进化地位及所编码蛋白的功能均有一定意义。     

中国人HCV基因组NS3区c33-c抗原基因cDNA克隆和在大肠杆菌中表达

本文通过逆转录(RT)-聚合酶链式反应(PCR)从一份来自山东省泰安市的丙型肝炎病毒(HCV)RNA打点杂交阳性的中国人血清中扩增并克隆到一段约850bp的HCV基因组NS3区c33-c抗原基因,测定该基因的全序列后发现,中国人HCV泰安分离株与HCV Ⅰ型株HCV-US和Ⅱ型株HCV-BK在该基因区段的核苷酸/氨基酸序列同源性分别为79.2％/91.3％和91.3％/93.9％。利用原核高效表达载体pBV220在大肠杆菌中有效地表达了非融合的c33-c重组蛋白,通过免疫筛选法及Western印迹法对约占菌体可溶性蛋白14％的表达产物进行了鉴定。采用Triton X-100和尿素处理表达产物后再进行离子交换层析,纯化得到可用于检测HCV血清抗体的c33-c抗原。利用该抗原与HCV核壳蛋白区的分支状合成肽一起组装成中国第二代免疫酶法(E1A)测定HCV抗体试剂盒,其检测特异性、灵敏度和精密性完全符合HCV诊断试剂的国家质控标准,已基本达到国际上第二代HCV E1A主流试剂的水平。     

THE VACCINIA VIRUS HindⅢ K FRAGMENT ENCODES A NOVEL PROTEIN BELONGING TO THE SERPIN SUPERFAMILY

The 1893-base pair nucleotide sequence of the EcoRⅠ-HindⅢ fragment of vaccinia virus Tiantan strain HindⅢ K clone is determined by the dideoxy chain termination method. A search in the NBRF protein sequence database using FASTA and other microcomputer programs reveals that several proteins belonging to the serpin (serine protease inhibitor) superfamily have striking similarities to tho protein encoded by the HindⅢ Kl ORF. On the basis of the dot-matrix analysis and sequence alignment, the Kl-encoded protein is shown as a novel member of the serpin superfamily. The putative reactive site and switch sequence of this novel serpin are then compared with those of other serpins. The probable evolutionary and possible functional relationships are discussed.     

人嗜中性白细胞活化蛋白-1/白细胞介素-8基因的半化学半酶促合成及在大肠杆菌的表达

本文采用半化学半酶促法合成了人嗜中性白细胞活化蛋白-1/白细胞介素-8(NAP-1/IL-8)的全基因,并在大肠杆菌系统中利用P_RP_1串联启动子进行了温控型的高效表达,通过SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表明,在摇瓶培养的大肠杆菌中,NAP-1/IL-8的表达量可占菌体可溶性蛋白的18.5％,而在实验室规模的发酵试验中,其表达量则占可溶性蛋白的10.9％.经凝胶过滤层析和阳离子交换层析两个简单的纯化步骤,得到SDS-PAGE纯的重组NAP-1/IL-8,采用琼脂糖平板法进行测定,表明所得NAP-1/IL-8纯品在<10ng/ml的水平上显示出强烈的嗜中性白细胞趋化活性,家兔皮肤试验的结果显示,重组人NAP-1/IL-8可在家兔体内引起早发型超敏反应,采用Edman降解法测定重组人NAP-1/IL-8纯品N末端36个氨基酸的序列,与天然人NAP-1/IL-8的成熟分子完全符合。