带肝细胞受体结合区的乙型肝炎病毒表面抗原蛋白性质的研究——形成表面抗原颗粒和在不同细胞中的分泌

我们用定向缺失突变和体外重组法构建了含有肝细胞受体结合区的乙型肝炎病毒(HBV)表面抗原主蛋白(S蛋白)基因。该基因在猴肾细胞COS-M6中的表达产物(S309蛋白)能形成表面抗原(HBsAg)颗粒并分泌出细胞。它在细胞和培养液中稳定,并能分别被抗HBsAg和抗preSl区的抗血清所沉淀。CsCl密度梯度分析显示S309蛋白形成颗粒的密度比S蛋白略大(1.25g/ml)。S309蛋白在肝癌细胞株中容易分泌,分泌量和S蛋白相近。但它在COS-M6细胞中的分泌和肝细胞株相差悬殊,分泌量只有10％左右,而且在培养液中出现较迟。和S蛋白的同时表达则能明显地提高它的分泌效率。     

促进动物生长的新型基因工程疫苗研究

将动物生长抑素(ss)基因融合到乙型肝炎病毒表面抗原(HBsAg)基因3′末端,构建融合基因重组痘苗病毒。重组病毒(vv-HBs/ss)能表达HBsAg杂合颗粒,并将ss抗原决定簇暴露在颗粒表面。以vv-HBs/ss作为活载体苗免疫家兔,能刺激产生ss抗体;免疫大鼠,能促进生长。     

THE CLONED SALI FRAGMENTS OF Bombyx mori NUCLEAR POLYHEDROSIS VIRUS DNA

Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA has been cleaved with restriction endonuclease SalI into 29 fragments of different sizes. The range of the molecular weight of these fragments as detected by electrophoresis on agarose gel is from 0.70 Kb to 10.0Kb. 24 SalI fragments were cloned in plasmid pBR322. The sum of the molecular weight of cloned BmNPV DNA fragments accounts for about 80% of the virus genome DNA.     

adr亚型乙型肝炎病毒表面抗原基因在大肠杆菌中的表达

利用adr亚型乙型肝炎病毒(HBV)DNA克隆株,组建表面抗原(HBsAg)基因表达质粒。用竞争性抑制放射免疫分析方法和聚丙烯酰胺——SDS凝胶电泳检测表达产物,获得了含HBsAg的杂合多肽。     

CHEMICAL SYNTHESIS AND CLONING OF SECRETIN GENE

A DNA duplex coding for the 27 amino acids of secretin has been synthesized and cloned. Indesigning the sequence of the gene, computer analysis has been applied. The following factors have beenconsidered: selection of codon usage in favour of expression in yeast; design of various sites useful ingene cloning, gene modification and expressed product purification; avoiding the repeat sequences whichmay interfere in the ligation of the synthetic fragments. The synthesis involved preparation of 12 oligo-deoxyribonucleotides (12-mer to 24-mer in length) by phosphate triester and phosphite triester method,purification by polyacrylamide gel electrophoresis (PAGE). A new plasmid pWS1 was constructed by inser-tion of the enzymatic ligated gene fragment into plasmid pWR13.     

A cDNA CLONE WITH ANTIONCOGENE ACTIVITY

A cDNA clone, p14--6, which has an antioncogene activity on the v--Ki--Ras oncogene-transformed malignant cell line DT, was found. This clone was recovered from the revertantR14 cells, which had been isolated by transfections of DT cells with a normal human fibro-blast cDNA library cloned in pcD2, an Okayama-Berg vector. When transfected into DT cells,p14--6 clone gave rise to phenotypical flat reversion in 5--15% of DT transfectant colo-nies. The p14--6--transfected flat cell line, RR, was proven to be a true revertant with signif-icantly reduced malignancy by in ritro and in riro malignancy tests. All other clones recov-ered from R14 cells were unable to cause this reversion. Molecular hybridizations showedthat the p14--6 was inserted into RR genome as tandem repeats, and no structural changewas found in the D--Ki--Ras oncogene in RR genome. These facts suggest that the antioncogeneactivity of the p14--6 clone on the DT cells may be exerted through expression of thecDNA contained in this clone. Possib     

THE POLYHEDRIN GENE OF Bombyx mori NUCLEAR POLYHEDROSIS VIRUS

Cloned SalI fragments of Bombyx mori nuclear polyhedrosis virus (BmSNPV) DNA were screened with the polyhedrin gene of Autographa californica nuclear polyhedrosis virus as a probe. One positive clone, pBN61, with an insert of 1.65 Kb, was obtained. The Hind-Ⅲ, HpaⅡ and AluⅠ maps of the insert were constructed. Part of its nucleotide sequence has been determined. The 46 amino acid sequence, as determined from the nucleotide sequence, was compared with the reported sequence of BmSNPV polyhedrin. Only ono amino acid difference has been found. It is likely that clone pBN61 contains the whole BmSNPV polyhedrin gene.     

具有抗癌基因活性的一个cDNA克隆

本文发现了一个对受v-Ki-Ras基因恶性转化的小鼠成纤维细胞株DT具有抗癌基因活性的cDNA克隆p14-6。该cDNA克隆系从用正常人cDNA库转染DT造成的回复突变细胞株R14中回收得到的。用p14-6再次转染DT细胞株时,可使约5—15％的DT细胞发生形态回复突变。p14-6所造成的形态回复细胞系RR经体内和体外的恶性检验,证明其确为恶性程度较DT有显著降低的回复突变系。从R14中回收的其它cDNA克隆均不能产生此种回复突变。分子杂交显示p14-6以线形多聚体整合入RR基因组;v-Ki-Ras基因结构无可检出的变化。这些事实提示cDNA克隆p14-6对DT细胞的抗癌基因作用可能是由其cDNA的表达所引起。 对部分DT细胞被p14-6质粒转染后恶性不变的可能原因进行了讨论。     

THE COMPLETE NUCLEOTIDE SEQUENCE OF THE CLONED DNA OF HEPATITIS B VIRUS SUBTYPE adr IN pADR-1

The complete nucleotide sequence of the cloned hepatitis B virus DNA subtype adr in pADR-1 wasdetermined by Maxam and Gilbert's method. It is 3215 base pairs in size, which is 27 bp longer thanthe sequence of the adr pHBr330, as reported by Ono et al. The nucleotide difference between pADR-1and adr pHBr330 is about 2% while those between pADR-1 and adw as well as ayw are 9.3% and 9.7%respectively. In this paper, the heterogeneity and homogeneity of the S gene, the C gene and the othercoding regions in pADR-1 and in the other subtypes are compared and discussed.     

A NEW SYSTEM FOR THE SYNTHESIS OF HIGH LEVELS OF HBsAg SEQUENCE IN Escherichia coli

Using a system to study promoter activity, we have obtained a promoter fragment from E. coli chromosomal DNA. The HBsAg geno under the control of this promoter could be expressed in E. coli. The expression products are isolated and purified by means of a column of anti-HBs cross-linked to Sepharose 4 B. The pure products are characterized through the double diffusion method on 0.6% agarose and polyacrylamido-SDS gel electrophoresis. The synthesis of high levels of HBsAg sequences in E. coli is confirmed.