Cloning and sequence analysis of human adiponectin gene

Liangdi XIE; Jie HUANG

ABSTRACT

BACKGROUND:

AdiponecUn (apM1) has been a new target for gene therapy of ischemic diseases; apM1 gene cloning is the key of successful apM1 gene therapy. There are two main gene cloning

methods:

directional cloning and T-A cloning method. Directional cloning method is complicated, while the T-A cloning method is relatively simpler, with higher successful rate.

OBJECTIVE:

T-A cloning of apM1 gene coding region was applied to verify the sequence in comparison with GenBank. DESIGN, TIME AND

SETTING:

The gene verification experiment was performed at the laboratories of Fujian Hypertension Research Institute which is in The First Affiliated Hospital of Fujian Medical University and Academy of Integrative Medicine which is in Fujian University of Traditional Chinese Medicine, from June 2006 to December 2008. MATERIALS Adipose tissue sample of omental fat pad was obtained from a surgical patient in the First Affiliated Hospital of Fujian Medical University. Trizol produced by Invitrogen company; M-,-MLV, Gel Extract Kit produced by PROMEGA company; Taq enzyme produced by TIANGEN company; Restriction Endonucleases BamH Ⅰ and Sal Ⅰ, pMD18-T Vector produced by TAKARAcompany were used in this study.

METHODS:

Total mRNA was extracted from human greater omentum adipose tissue. The coding region of human apM1 (hapM1) gene was amplified by RT-PCR. The coding region of hapM1 gene was cloned into pMD18-T vector .The recombinant plasmid was identified with restriction enzyme digestion analysis and nucleotide sequencing. MAIN OUTCOME

MEASURES:

The electrophorasis verification of the recombinant plasmids coding target gene using double enzyme digestion, comparison of the similarity between the sequences of the plasmids and hapM1 in GenBank.

RESULTS:

Sense and antisense coding region of hapM1 gene were cloned. The sequencing results showed that the sequences of the cloned DNA were completely identical to that of hapM1 in GenBank.

CONCLUSION:

The coding region of hapM1 gene was successfully cloned.