带有EGF受体干扰序列的γ干扰素新分子抗肿瘤细胞增殖活性的提高

利用基因工程技术,将天然IFN-γ分子的N端132个氨基酸与带有上皮生长因子(EGF)样多肽C端保守序列融合,构建了干扰素重组体IFN-γ132PGIX_(16).其中X_(16)由EGF样多肽C端16个氨基酸组成,PGI为连接肽序列。将此重组体基因插入表达载体pBV220P_RP_L串连启动子下游,转化大肠杆菌DH5α,在cIts857基因的调节下,通过升温诱导表达.表达产物与其母体天然IFN-γ相比,不仅保持了较高的抗病毒活性,抗病毒滴度达530MU/L菌液,而且明显地提高了抗肿瘤细胞增殖活性.在EGF存在的条件下,干扰素重组体的抗鼻咽癌细胞CNE-2增殖活性明显高于其母体分子(p<0.01).提示重组体分子IFN-γ132PGIX_(16)同时具有EGF受体干扰活性.     

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。     

一种αⅠ型干扰素基因新变种的发现和鉴定

本文从一名中国汉族正常人胎肝细胞染色体DNA中,应用PCR方法两次获得长为520 bp的人α型干扰素基因,核苷酸序列测定表明,两次扩增产物的DNA序列完全相同,与过去分离克隆的IFN-αI和IFN-αD基因相比较,其第410位和第541位核苷酸分别为C和G,由此推测它编码的IFN成熟肽第114位和第158位氨基酸应为Ala和Val,其余位点则与IFN-αD和IFN-αI完全相同.我们建议将IFN—αD,IFN—αI和我们分离的IFN-αI/158V基因分别命名为IFN-αIa,IFN-αIb和IFN-alc基因.     

我国人乳头瘤病毒6型新变种基因组克隆及L1晚期结构基因在大肠杆菌中表达

本文从一例中国妇女外阴尖锐湿疣组织标本中提取DNA,BamHI酶切,以pAT153为载体,成功地克隆了我国人乳头瘤病毒6型(HPV-6BV)基因组.经South-ern杂交,酶谱及部分DNA序列分析,发现该克隆的HPV6基因组有明显变异,可视为我国发现的第一个HPV6的新变种.本文进一步分离了HPV6BV的L1基因,以pUR288为载体,在大肠杆菌中表达了β-半乳糖苷酶/HPV6BVL1N融合蛋白.经免疫转印试验证明,它既能与β-半乳糖苷酶抗血清反应,又能与HPV抗体反应,具有融合前各自的抗体亲和活性.该蛋白可用作诊断试剂及流行病学研究.     

MOLECULAR CLONING OF A NEW VARIANT OF HUMAN PAPILLOMAVIRUS TYPE 6 ISOLATED FROM A CHINESE WOMAN AND EXPRESSION OF ITS L1 GENE IN E. coli——MOLECULAR CLONING & EXPRESSION OF HPV6 GENE

A human papillomavirus genome DNA of 7.9 kb from a Chinese woman with genital condyloma acuminata was cloned in Bam HI site of pAT153. According to the results obtained from Southern blotting, restriction mapping as well as partial DNA sequencing, the isolated genome (HPV6BV) had obvious variance and was referred to as a new variant of HPV6 found in China the first time. HPV6BV L1 gene was successfully expressed in E. coli as a fusion protein with pUR288. The β-galactosidase/L1 fusion protein reacted with both β-galactosidase antiserum and HPV antibody using Western blot technique. The E. coli-produced fusion protein, possessing HPV antigenicity, may provide a reagent for clinical diagnosis and epidemiological survey.     

中国人ω1型干扰素基因的克隆、一级结构测定以及在大肠杆菌中的表达

采用聚合酶链反应(PCR)方法,从中国正常胎儿肝细胞染色体DNA中分离并克隆了人ω1型干扰素基因,测定了核苷酸全序列,表明原有争议的第88位密码子为GGA的核苷酸序列是正确的,因此该氨基酸为Gly。随后又利用PCR技术将所获的ω1型干扰素基因原始克隆改造成适于在大肠杆菌中表达非融合蛋白的结构形式,并以pBV220为载体在P_RP_L串联启动子控制下进行温控表达。表达产物的抗病毒活性达6.5×10~7单位/升菌液(O. D_600=0.75)。IFN-ω1不仅具有很高的抗病毒活性,同时在结构上与动物滋养层蛋白高度相似,而后者又是作为母体妊娠识别信号存在于多种动物体内的抗黄体退化蛋白,因此本文获得的这—ω1型干扰素基因及重组蛋白在理论和实际上都有进一步研究的价值。     

Cloning, Sequencing and Expression in E. coli of Interferon-ω1 Gene

Human interferon ω1 (huIFN-ω1) gene was isolated and cloned from chromosome DNA derived from a Chinese fetal liver via polymerase chain reaction (PCR). By determining its nucleotide sequence we proved that the 88th codon should be GGA, coding for Gly. After engineering the original IFN-ω1 gene clone to a form that may be expressed as a nonfused protein, we also took the IFN-ω1 gene under the control of the PRPL promoter with an expression vector pBV220 in E. coli. The antivirus activity of the recombinant IFN-ω1 is about 6.5×10~7 units/L CULTURE (OD_(600)=0.75). Since IFN-ω1 not only has antivirus activity but also shows considerably high homology with animal trophoblast proteins which have been proved antiluteolysins as a maternal recognition signal for pregnancy, we believe that study on it will be practically and theoretically significant.     

人α-干扰素cDNA在转化的烟草植株中表达

本试验成功地将人的α-干扰素cDNA导入烟草细胞中,并得到了产生活性干扰素的转化烟草植株。将人的α-干扰素的cDNA通过平头末端连接的方法插入到植物表达质粒pAP2034中的T-DNA转录子1的启动子及其转录子7的加尾信号间的BamHI位点,构建了重组质粒piG3031,将此重组质粒导入Ti质粒载体pGV3850的T区,利用农杆菌感染叶盘的方法转化普通烟草1551,得到了抗卡那霉素的转化植株。DNA的Southern杂交表明:人的α-干扰素基因随同T-DNA一起整合进了烟草基因组中,新霉素磷酸转移酶Ⅱ(NPTⅡ)的检测表明:转化植株之所以能抗卡那霉素是因为NPTⅡ酶基因表达的缘故。而干扰素的生物活性检测的结果说明转化植株能产生有活性的α-干扰素。