Neijiang Pig Interleukin-2 Cloning And Prokaryotic Expression

Interleukin-2 (IL-2) is a key component for cellular immunity in animal. It plays important roles in the regulation of immune system. Medical science has particular interests in the research of IL-2. However, only few researches have been reported for IL-2 expression in prokaryota. The objectives of this study were to clone the interleukin-2 of Neijang pig, to determine IL-2 DNA sequence, to express IL-2 genes in prokaryote, to determine the biological activity of protein expression in the prokaryote, finally to obtain a new strain expressing porcine IL-2. Based on the nucleotide sequence of porcine IL-2 published (X58428), we designed and synthesized a pair of primers. Total cellular RNA, isolated from con-stimulated peripheral blood lymphocytes of Neijiang pig, was used as template to generate complementary DNA via reverse transcription. The 540bp DNA fragments were amplified by polymerase chain reaction (PCR), and successfully cloned into T-vector. DNA sequence analysis confirmed that the DNA fragment was the gene for IL-2 expression in Neijiang pig. This fragment included the entire ORF of the Neijiang pig IL-2, and encoded the 155 amino acids of the IL-2 protein. DNA sequence analysis showed that the cloned sequence had 99.6% similarity with those published on the Genebank for pig, only had two replacements at sites 245 (Aâ†'T) and 257 (Tâ†'A). Using the restriction endonuclease sites of EcoRI and HindIII from PET-32a(+), the cloned fragment was inserted to the plasmid to construct prokaryotic expression plasmid PET-32a(+)-IL-2. This recombinant plasmid was transformed into E.coli Bl21(DE3) to establish a new IL-2 expression system. A recombinant syncretic protein with M.W about 37KD was identified in this system. Biological activity of the expressed recombinant protein was investigated with MTT method and significant result was observed when ConA-stimulated Neijiang peripheral blood lymphocytes cells were used as the response cells. Stimulation time, MTT concentrate, and MTT reaction time in the MTT method and other parameters were tested to optimize the conditions for measurement of the activity of the recombinant protein.In this study, we successfully established a method to construct a new strain of prokaryote that can expresses porcine IL-2. This strain may have potential commercial applications in swine disease prevention.