Cloning and sequence analysis of Wild Argali short palate, lung and nasal epithelium clone 1 cDNA.
OBJECTIVE
Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 (SPLUNC1) cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1.


METHODS
The complete sequence of Wild ArgaliSPLUNC1cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed inPichia pastoris(P. pastoris) GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by Ni2+chelate affinity chromatography. The test of effect of the protein onMycoplasma ovipneumoniae(MO) was performed with real-time polymerase chain reaction.


RESULTS
The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA fromOvis aries(accession no. NP_001288334.1),Capra hircus(accession no. XP_005688516.1),Pantholops hodgsonii(accession no. XP_005979709.1),Bos taurus(accession no. NP_776851.1),Felis catus(accession no. XP_006929910.1),Homo sapiens(accession no. NP_001230122.1),Sus scrofa(accession no. NP_001005727.1),Chinchilla lanigera(accession no. NP_001269294.1),Mus musculus(accession no. NP_035256.2), andRattus norvegicus(accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant ofP. pastoris, and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01).


CONCLUSIONS
The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.
College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003, China.|College of Life Science, South China Agricultural University, Guangzhou, Guangdong 510000, China.
Wen
W
Shen
College of Life Science, Shihezi University, Shihezi, Xinjiang 832003, China.
Kaili
K
Chen
College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003, China.
Yanming
Y
Sun
College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003, China.
Haiying
H
Guo
College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003, China.
Dongmei
D
Chen
College of Life Science, South China Agricultural University, Guangzhou, Guangdong 510000, China.
Yang
Y
Cao
Activity Detection; Argali; Clone; Expression; Lung and Nasal Epithelium Clone 1 (SPLUNC1) cDNA; Sequencing; Short Palate