Cloning And Expression Of Ovine CCL28 And Granulysin In Vitro

CC chemokine ligand 28 (CCL28), also called mucosae-associated epithelial chemokine (MEC), is the member of the CC chemokine subfamily which was discovered recently and expressed in most mucosal tissues of human and mouse which include the salivary gland, mammary gland, small and large intestines and trachea, where it is predominantly produced by epithelial cells. CCL28 play a essential role in homing of IgA antibody secreting cell (IgA-ASCs), and CCL28 also has a broad-spectrum antimicrobial activity.Granulysin, a antimicrobial protein expressed in Cytolytic T Lymphocytes (CTL) and Natural Killer cells (NK cells), is a member of the saposin-like protein (SAPLIP) family of lipid binding proteins and the only one antimicrobial effector molecule which was identified in CTL endochylema at present. Granulysin can colocalizes in human CTL and NK cells with perforin and granzymes. Its function is similar to broad-spectrum antibiotics against both Gram-positive and Gram-negative bacteria, fungi, and parasites. Granulysin also kills human tumor cells.To investigate if CCL28 and Granulysin play same roles in the sheep, we cloned ovine CCL28 and Granulysin and obtained cognate fusion protein. The study was divided into two series.â… . Cloning and expression of ovine CCL28 in vitro. A pair of cloning primers were designed according to the cloning principle of homologous sequence. Total RNA extracted from mucosal tissue of ovine colon was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into Ecol. JM109 competent cells. The positive clone was identified and the sequence was sequenced and analyzed. Then, a pair of expression primers were designed according to the cloned sequence. A fragment of ovine CCL28 cDNA containing BamHI/XhoI was amplified from recombinant vector by PCR. The fragment digested by BamHI/XhoI was subcloned into pGEX-4T-1 vector to construct a recombinant prokaryotic expression vector, pGEX-CCL28. Subsequently, the recombinant vector was transformed into E.coli BL2l competent cells. The fusion protein induced by IPTG was analyzed by SDS-PAGE. The cloned sequences containing the open reading frame of ovine CCL28 consist of 444bp, and ORF is 387bp and encodes 129 amino acids. Identity analysis showed that the ovine CCL28 nucleotide sequences shared 76.4%, 61.4%, 84.3% and 92.9% homology with that of human, mouse, pig and cattle, the deduced amino acid sequences shared 76%, 63%, 84% and 93% homology with that of human, mouse, pig and cattle. The signal peptide is 1-24aa and the domain SCY is 27-88aa, which reveals that the structural feature is consistent with human, mouse pig and cattle. The prokaryotic expression pGEX-CCL28 was successfully constructed. The fusion protein expressed in E.coli BL21 was about 39ku. It provides the experiment basis for further researching its biological function.â…¡. Cloning and expression of ovine Granulysin in vitro. A pair of cloning primers were designed based on the in silico sequence information. Total RNA extracted from mucosal tissue of ovine ileum was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into Ecol. JM109 competent cells. The positive clone was identified and the sequence was sequenced and analyzed. Then, a pair of expression primers were designed according to the cloned sequence. A fragment of ovine Granulysin cDNA containing BamHI/XhoI was amplified from recombinant vector by PCR. The fragment digested by BamHI/XhoI was subcloned into pGEX-4T-1 vector to construct a recombinant prokaryotic expression vector, pGEX- Granulysin. Subsequently, the recombinant vector was transformed into E.coli BL2l competent cells. The fusion protein induced by IPTG was analyzed by SDS-PAGE. The cloned sequence containing the open reading frame of ovine Granulysin consists of 509bp, and ORF is 438bp and encodes 146 amino acids. Identity analysis showed that the ovine Granulysin nucleotide sequence shared 56.2% and 87.2% homology with that of human and cattle, the predicted amino acid shared 38% and 75% homology with that of human and cattle. The signal peptide is 1-22aa and the domain SapB is 64-138aa, which reveals that the structural feature is consistent with human and cattle. The prokaryotic expression pGEX-Granulysin was successfully constructed. The fusion protein expressed in E.coli BL21 was about 41ku. It provides the experiment basis for further researching its biological function.