Biomedical subjects

J H Chai

Publications and source records attributed to J H Chai.

4 recordsLinked to original sources

PubMed1992

Expression of a partial synthetic human TNF cDNA in E. coli.

A recombinant human tumour necrosis factor (rhTNF) cDNA was constructed. The TNF gene was isolated from a human genomic gene library. There are four exons in the TNF gene. The fourth exon codes for 140 amino acids of the TNF matured protein which is composed of 157 amino acids. A major portion of the fourth exon was isolated and then ligated to a synthesized DNA fragment coding for the remaining amino acids. The partial synthetic hTNF (rhTNF) cDNA thus generated was subcloned into a vector and successfully expressed in E. coli. 5-1 fermentator was used to produce rhTNF. About 20 g (wet weight) of bacterial pellet per liter medium and 10(6)-10(7) units of cytotoxicity to L929 cells per milliliter medium were obtained. rhTNF was purified by HPLC and dried with a freeze dryer. rhTNF with a purity of about 95% in the form of white powder was obtained. The sequence of ten amino acids at the amino terminus of the rhTNF was determined. The result showed that it was identical with that of the natural human TNF.

Base Sequence↗

PubMed1990

Molecular cloning and restriction mapping of human lymphotoxin gene.

In order to clone the human lymphotoxin (HuLT) gene, we practiced a concise and time-saving method: homologous recombination in vivo (1). By using the mouse lymphotoxin (MuLT) cDNA (1.3 kb) as a probe, we isolated the HuLT gene from a human genomic library which was constructed with cosmid pcos2EMBL as a vector. After linearization, the recombinant cosmid was partially digested with BamHI, EcoRI, PstI, and PvuII respectively, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence (2). The physical map of HuLT gene was made by this method.

Blotting, Southern↗

PubMed1990

[The selective isolation of cosmid clones by homologous recombination in Escherichia coli--a cosmid clone containing t complex linkage DNA sequence of mouse was isolated].

A procedure for the selective isolation of specific cosmid clones by homologous recombination between cosmid clones of genomic library and a probe DNA sequence cloned in a plasmid in vivo has been developed. The cosmid library was constructed in a rec- host cell strain and packaged into phage particles in vivo. The rec+ host cells containing a DNA sequence used as selection probe cloned in the pUC plasmid were infected by packaged cosmid phage particles. There is no homology between cosmid and the plasmid vectors. After a period of 1-3 hr. for the recombination to take place, the probe plasmids were integrated into cosmid, in which the DNA sequence are homologous with the probe, by homologous recombination. The cosmids are then packaged in vivo and transferred into a rec- cell strain. The specific cosmid clones were selected by double antibiotic resistance carried by both vectors. The probe plasmid can be excised by lambda excision enzyme by using superinfection with red+ phage. After packaging in vivo, these cosmid revertants can be identified on Xgal plate. A cosmid clone containing the t complex linkage DNA sequence of mouse was selected by using the procedure above with a probe derived from microdissected metaphase chromosome.

Animals↗

PubMed1985

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Animals↗