Abstract

Background: Glucosidase II is an endoplasmic reticulum heterodimer enzyme, a neutral glucosidase, involved in the trading and folding of newly synthesized glycoproteins in the endoplasmic reticulum. The destruction of GANAB leads to the accumulation of misfolded glycoproteins and the induction of unfolded protein responses. GANAB is also a key regulator of glycosylation. The encoded GANAB protein has an increased expression level in lung tumor tissues, but its function in lung cancer is not yet clear. The cloned sequence is the mutant gene of GANAB gene (NM_198334.3) in the Genbank database, and its role in lung cancer.

Method: In this study, the GANAB gene was obtained by homologous cloning, and the correct gene sequence was verified by sequencing by bioinformatics analysis; and the cDNA of the GANAB gene was cloned by RT-PCR. Aiming at high expression level, use the T base of pMD19-T Vector and the A base of the RT-PCR amplified cDNA to match the GANAB cDNA, and use the pcDNA3.1 plasmid to construct a high expression GANAB plasmid, and then use the sensory cell plasmid to purify. Western blot analysis was used to confirm the expression of the protein.

Result: The cDNA fragments of 1.8kbp and 1.0kbp were isolated by RT-PCR experiment. Use pMD19-T Vector to connect the GANAB cDNA fragment to construct a 5.5kbp cDNA sequence and use the pcDNA3.1 plasmid to construct a GANAB plasmid to obtain an 8.2kbp cDNA sequence. Western blot analysis confirmed the expression of the protein.

Conclusion: In this study, we cloned the GANAB gene in A549 cells and successfully introduced the forced expression into A549 cells. The cloning and protein expression of GANAB gene are of great significance to the study of the key effects of lung cancer adhesion, invasion and metastasis.