Construction Of CDNA Library From Ear Tissue Of Luxi Yellow Cattle And Tβ4 Gene Cloning And Expression

Luxi Yellow Cattle is one of important endangered species in China, and the construction of cDNA library of Luxi Yellow Cattle is very important to save the genetic resources as well as to study the gene functions.The total RAN were extracted from ear fibroblast cells of Luxi Yellow Cattle, and the ear tissue cDNA library were constructed by using SMARTTM technique in this study. The identification result showed that the titers of unamplified and amplified cDNA library were 2.88×106pfu/mL and 1.33×1010pfu/mL, respectively. 96 clones were randomly selected from the amplified library and analyzed by PCR electrophoretic analysis. The results showed that the recombination rate was 92.7% and the average size of insert fragments was 1.0kb.And then Thymosin beta 4 gene was cloned and its prokaryotic expression vector, pGEX-4T-1-Tβ4, was successfully constructed. The pGEX-4T-1-Tβ4 fusion protein was detected by using SDS-PAGE following the induced expression of different gradient concentration of IPTG. The results showed that the expression strip of Tβ4 fusion protein was appeared at 32KD compared to the control, indicated the Tβ4 gene was expressed in BL21 successfully. Therefore, these experimental results would establish the foundation for further study of biological functions of Tβ4 gene.