Method for establishing eukaryotic expression cell line of CD36 mutant gene that encodes CD36 deficiency

US10711046B2 · kind B2 · utility

Assignee

• NAN-NING INSTITUTE OF TRANSFUSION MEDICINE · CN

Inventors

• Guoguang WU · Nanning, CN
• Lilan LI · Nanning, CN
• Lihong Jiang · Jilin, CN
• Haiyan Li · Longbeilingcun, CN
• Jierun CHEN · Nanning, CN

Key dates

• Filing date: Nov 12, 2018
• Grant date: Jul 14, 2020
• Expiry date: Nov 12, 2038

Classification

• Technology area (CPC Y): Emerging Cross-Sectional Technologies
• CPC primary: Y02A50/30
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A method for establishing eukaryotic expression cell line of CD36 mutant gene that encodes CD36 deficiency, the method including: (1) extracting total RNA from a whole blood sample derived from a CD36-deficient individual, and amplifying a coding sequence (CDS) in CD36 mRNA, to obtain a cDNA sequence fragment of the mutant CD36 gene; (2) splicing and amplifying the mutant CD36 gene and the EGFP fluorescent gene by SOE-PCR (Gene Splicing By Overlap Extension PCR) using four forward and reverse primers, to obtain a mutant gene fragment of MT-CD36-EGFP; (3) constructing and amplifying a MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector including the mutant CD36 gene and the EGFP fluorescent gene; (4) transfecting the MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector into the CHO-K1 cell line by using virus-mediated transfection of eukaryotic cells.