Clone And Expression Of C.mongolicus Ob Gene

In this study, Culter mongolicus (Basilewsky) were taken from South Lake fishing ground; the length and weight of male and female fish were about 23.1cm, 151.3g and 25.2cm, 155.6g each. Tissues such as adipose, liver, heart, spleen, muscle, brain, ovary, kidney, spermary had been obtained from the double fish and deserved in the refrigerator at -70 ℃ after cleaned by distilled water. Each tissue's total RNA were extracted by one step method and the extracted total RNAs were identified by Eppendorf Bio-photometer. All the total RNA that ratios of OD₂80/OD²60 were from 1.8 to 2.0 were diluted into 0.5μg/μL. The primers were designed according to the consensus region of human and mouse ob genes and were employed to perform RT-PCR. The primer was included EcoR I and Hindâ…¢ enzyme cut site at sequence. The product of RT-PCR and pBluescript SK(+) vector were both digested by EcoR â…  and Hind â…¢, then linked and transformed into DH5 α competent cell. The recombined plasmid was extracted and then identified by PCR and double enzyme digestion. The positive recombined plasmid pSK-ob was sequenced and got a 438bp coding region DNA sequence. Comparative to human, pig and mouse in the nucleotide sequences of ob genes, the homology was 82.9%, 84.0%, and 99.1% respectively. The nucleotide sequences of ob genes were very conservative among the four animal species. In comparison with human, pig and mouse in the Leptin amino acid sequence the homology of amino acid sequences were 80.8%, 78.8%, 95.9% in order. The result of ob gene tissue express specificity by RT-PCR method indicated that ob gene was expressed mainly in adipose and liver, less in heart, spleen, muscle, brain, ovary, slightly in kidney and none in spermary and intestines. The ob gene was cloned to the pET-28a, and transformed into BL21 (DE3), then induced to expression. The expression product was analysed by SDS-PAGE and showed that the protein molecular weight was about 20kD, whereas fusion protein molecular weight was 4KD, so the remaining protein molecular weight was 16KD that was as large as we hoped.