Construction of expressive plasmid for full-length cDNA encoding human islet cell autoantigen 69kD protein

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Author:
Huang Naiping()
Cai Jun()
Wu Rui()
()
Journal Title:
CHINESE JOURNAL OF DIABETES
Issue:
Volume 8, Issue 02, 2000
DOI:
Key Word:
Islet Autoantigen Molecular cloning Type 1 diabetes

Abstract: Objective By means of the gene engineering technique, it would be possible to construct an expressive plasmid for ICA cDNA for providing the diagnosis kits for type 1 diabetes mellitus.Methods Total RNA were extracted from the pancreatic cells of Caucasian and the insulinoma cells of Chinese and cDNA of islet cell autoantigen 69kD genes (ICA69) were synthesized by superscript reverse transcriptase and polymerase chain reaction (PCR). After determining its nucleotide sequence by the dideoxy chain termination method, the encoding fragment of hICA69 gene was demonstrated to be composed of 1449bp nucleotides which would then be inserted into the pGEX 2T expression plasmids.Results It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5′ terminal multi cloning site and recombinant fragment after the analysis of the nucleotide sequence.Conclusion This investigation would be able to lay a foundation for hICA69 gene expression and clinical application of expression products.

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