[Cloning, sequencing and prokaryotic expression of cDNAs for the antifreeze protein family from the beetle Tenebrio molitor].
The partial cDNA sequence coding for the antifreeze proteins in the Tenebrio molitor was obtained by RT-PCR. Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze proteins. The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E. coli BL21 to induce a GST fusion protein by IPTG. SDS-PAGE of the fusion protein demonstrated that the antifreeze protein migrated at a size of 38 kDa. The immunization was performed by intra-muscular injection of pCDNA3-tmafp-XJ430, and then antiserum was detected by ELISA. The titer of the antibody was 1:2,000. Western blotting analysis showed the antiserum was specific against the antifreeze protein. This finding could lead to further investigation of the properties and function of antifreeze proteins.
Liu
Zhong-Yuan
ZY
Key Laboratory of Molecular Biology, College of Life Science and Technology, Xinjiang University Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Urumqi 830046, China. lzy1168@163.com
Wang
Yun
Y
Lü
Guo-Dong
GD
Wang
Xian-Lei
XL
Zhang
Fu-Chun
FC
Ma
Ji
J
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics*; D004220:Disulfides; D005786:Gene Expression Regulation; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013712:Tenebrio
D021301:Antifreeze Proteins; D018076:DNA, Complementary; D004220:Disulfides; D019476:Insect Proteins; D011994:Recombinant Proteins