Expression of cloned beta-endorphin gene sequences by Escherichia coli

DNA coding for the opiate peptide beta-endorphin has been cloned into bacterial plasmids in such a way as to direct the synthesis of a hybrid beta-galactosidase/beta-endorphin protein. This hybrid protein can readily be cleaved in vitro to release biologically active beta-endorphin.     

Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA

A synthetic fowl plague virus (FPV) haemagglutinin gene has been cloned in bacteria and the complete sequence of the RNA gene deduced. It is 1,742 nucleotides long and the mRNA codes for 56.3 amino acids in an uninterrupted sequence. The nature of some of the important domains in the haemagglutinin has been established, and their structure is discussed in relation to their function. Extensive amino acid sequence homologies exist between FPV and human influenza haemagglutinins.     

重组人粒细胞集落刺激因子在Escherichia coli中的表达研究

根据人天然粒细胞集落刺激因子（Ｇ－ＣＳＦ）基因序列设计出引物,通过ＲＴ－ＰＣＲ从人外周血单个核细胞ｍＲＮＡ获得Ｇ－ＣＳＦｃＤＮＡ片段,将该片段与原核表达载体ｐＴ７构建成重组体,导入大肠杆菌后发现天然Ｇ－ＣＳＦｃＤＮＡ表达量并不理想,这可能是由于天然ｈＧ－ＣＳＦ５’端Ｇ＋Ｃ的比例过高,使转录后的ｍＲＡ很容易形成二级结构而影响翻译起始,因此在大肠杆菌中很难获得高效表达,根据密码子简并性原则,在不改变编码氨基酸顺序的前提下,通过重新设计ＰＣＲ引物,将ｈＧ－ＣＳＦ的前３个氨基酸密码子中的几个ＧＣ碱基作了改动,获得了新的ｃＤＮＡ突变体,将其与ＰＴ７的重组导入大肠杆菌,获得了高效表达。     

Cell-surface expression of influenza haemagglutinin from a cloned DNA copy of the RNA gene

By replacing either the eight early or the late genes of SV40 with a cloned copy of the influenza virus haemagglutinin gene we have constructed recombinant viruses which, in infected cells, express large quantities of haemagglutinin. This glycoprotein, over 10(8) molecules of which are produced per cell, is identical in molecular weight to authentic influenza virus haemagglutinin, accumulates at the cell surface and displays haemabsorbing activity.     

Antibodies from Escherichia coli

Use of Escherichia coli as an expression host has opened up new possibilities in antibody research and its applications. It greatly facilitates rational engineering and random mutagenesis.     

大肠杆菌pheA与tyrB基因的克隆与串联表达

为探讨用基因工程的手段改良苯丙氨酸的发酵菌株，采用聚合酶链反应（ＰＣＲ）的方法，从大肠杆菌总ＤＮＡ中克隆得到了编码苯丙氨酸合成途中的两个关键酶基因－即分枝酸变位酶（ＣＭ）／预苯酸脱水酶（ＰＤ）基因ｐｈｅＡ与苯丙氨酸转氨酶（ＰＡＴ）基因ｔｙｒＢ，在大肠杆菌中进行了这两个基因的单个和串联表达。ｐｈｅＡ和ｔｙｒＢ基因分别都能在λ噬菌体的ＰＲ启动子之后得到较大量的表达，在ＳＤＳ－ＰＡＧＥ上出现清晰的条带，     

Expression in Escherichia coli of hepatitis B virus DNA sequences cloned in plasmid pBR322.

Fragments of hepatitis B virus DNA isolated from Dane particles have been inserted into the Escherichia coli plasmid pBR322 and cloned. Cells carrying the hybrid plasmid synthesise antigenic material that reacts specifically with antisera to hepatitis B viral antigens.     

Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.     

产丁醇重组大肠杆菌的构建及初步发酵

将丙酮丁醇梭菌ATCC 824丁醇合成途径中分别编码硫解酶(THL)、3-羟基丁酰-CoA脱氢酶(HBD)、巴豆酸酶(CRT)、丁酰-CoA脱氢酶(BCD)和醛/醇脱氢酶(BDH)的关键基因thil、hbd、crt、bcd-etfB-etfA、adhE在大肠杆菌中进行重组表达,构建出3株产丁醇的重组大肠杆菌JM109-99d、DL1346 99d和AFP111-99d。选取摇瓶发酵产丁醇最高(0.52 g/L)的重组大肠杆菌DL1346-99d进行上罐发酵,丁醇产量可达1.07g/L。     

Phenylalanine biosynthesis in Brevibacterium lactofermentum using Escherichia coli genes pheA, aroG and tyrB

Genetic engineering technology to increase the production of L-phenylalanine was used in the study.Three genes encoding the key enzymes involved in the biosynthesis of L-phenylalanine were utilized, in which the gene aroG encodes 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase (DS); the gene pheA encodes bifunctional enzyme of chorisate mutase (CM) and prephenate dehydratase (PD); and the gene tyrb encodes aminotransferase (AT).The three genes were amplified by polymerase chain reaction (PCR) from the genome of the E. coli mutant strains resistant to fluro-DL-phenylalanine and inserted into the cloning vectors. Then, they were expressed in E. coli and Brevibacterium lactofermentum in a tandem arrangement. The expressed enzymes had high activities in the host cells.     

Deletion of immunoglobulin heavy chain genes from expressed allelic chromosome

We have studied the organization of immunoglobulin heavy-chain genes in a gamma 2b-chain (BALB/c allotype)-producing myeloma BKC F1 # 15 induced in a F1 mouse between C57BL and BALB/c. Southern blot hybridization studies using cloned mu, gamma 1 and gamma 2b-chain genes as probes demonstrate that the mu- and gamma 1-chain genes of the expressed chromosome are deleted while these genes of the unexpressed chromosome are retained. The gamma 2b-chain gene of the expressed allele is rearranged while that gene of the unexpressed allele seems unchanged, as do the gamma 2a-chain genes. These results support the allelic deletion mechanism in heavy-chain class switch and the order of H chain genes.