Cloning,expression And Activity Detection Of SP-A Gene In Sheep

The Bashibay sheep is a good local breed in Xinjiang.It has strong cold tolerance and disease resistance.In particular,the resistance to Mycoplasma ovipneumoniae.The crossbred sheep of Argali and Bashibay sheep inherited the genetic characteristics of large size and rapid growth and development of the sheep,but they were susceptible to Mycoplasma ovipneumoniae.The survival rate is low.Pulmonary surfactant-associated protein A(SP-A)is a hydrophilic glycoprotein,which belongs to the C-type lectin family and is an important innate immune defense molecule in the lung.It plays an important role in local defense and innate immune response of lung.Objective: To clone the full-length cDNA sequence of SP-A gene from Bashibay and Argali hybrid sheep,and compare it with other breeds of sheep and animals and express the SP-A gene of Bashibay and Argali hybrid sheep by eukaryotic expression.Finally,the liveness of recombinant pulmonary surfactant-associated protein A(rSP-A)on the growth of Mo in vitro was studied,which laid a foundation for the study of the function of r SP-A in vitro.Methods:(1)The clone of the full-length cDNA sequence of SP-A gene from Bashibay and Argali hybrid sheep: The samples of lung tissue of Bashibay and Argali hybrid sheep were collected,and the total RNA of two samples of lung tissue were extracted by Trizol method,specific primers were designed according to the SP-A gene sequence of sheep in GenBank.Then,the 3 'and 5' end sequences of SP-A gene of Bashibay sheep and Argali hybrid sheep were both cloned by RACE method.The full-length cDNA sequence of SP-A gene was obtained by sequencing and splicing.To analyze the homology of SP-A gene between Bashibay and Argali hybrid sheep and other animals.(2)Bioinformatics analysis of the full length of SP-A gene cDNA sequence of Bashibay and Argali hybrid sheep: Using online bioinformatics analysis tools and bioinformatics software to analyze the full-length cDNA sequence of SP-A gene of Bashibay and Argali hybrid sheep.It includes nucleotide sequence of SP-A gene,hydrophilic/hydrophobic,signal peptide of SP-A protein,coding amino acids,SP-A protein sub-cell localization,high-level structure of protein and phylogenetic tree analysis.(3)Eukaryotic expression and protein purification of SP-A gene in Bashibay and Argali hybrid sheep: The specific expression primers were designed according to the full-length cDNA sequence of SP-A gene of the two kinds of sheep cloned before.The ORF of SP-A gene of two kinds of sheep were amplified by RT-PCR method,and the recombinant expression plasmid pPIC9K-SP-A was constructed.The recombinant expression plasmid was transformed into Pichia pastoris GS115 by electric shock.The recombinant strainGS115/ pPIC9K-SP-A was induced to express rSP-A by carbinol and the target protein was detected by SDS-PAGE and Western Blotting method.The eukaryotic expression of rSP-A was purified by Ni-NTA Agar affinity chromatography.(4)The effect of Bashibay and Argali hybrid sheep's rSP-A on the growth of Mo:The effect of rSP-A on the growth of Mo in vitro was detected by plate colony counting method.Results and conclusion:(1)The results showed that the cDNA sequence of SP-A gene of Bashibay and Argali hybrid sheep are both 1962 bp,they have the same length of open reading frame which is 747 bp,both encode 248 amino acids,the 5 'non-coding region are both 111 bp,and the 3' non-coding region are both 1104 bp.The analysis of nucleotide differences showed that there was one nucleotide difference in the5'non-coding region of the two breeds and three differences in the 3'non-coding region and three in the open reading frame,which resulted in the change of two amino acids.The results of homology analysis showed that the nucleotide sequence of SP-A gene and the coding amino acids sequence of SP-A gene of Bashibay and Argali hybrid sheep had the highest homology with sheep.(2)The protein molecular weight of SP-A gene of Bashibay and Argali hybrid sheep encoded by SP-A gene was estimated by DNASTAR software to be 26.38 kDa and 26.39 kDa,and the isoelectric point of protein was 4.855 and 4.942,respectively.The results of hydrophilic/hydrophobic analysis showed that both sheep SP-A proteins had high hydrophilicity.The results of secondary and tertiary structure analysis showed that the secondary and tertiary structures of SP-A protein were mainly irregular curl,followed by ?-helix and ?-folding.There were one N-glycosylation site,five casein kinase ? phosphorylation sites,five acylation sites and one C-type lectin domain in both sheep SP-A proteins.The BLAST ratio of NCBI was used to predict the SP-A domain of Bashibay and Argali hybrid sheep.It contains collagen domain and CLECT domain.The results of protein signal peptide analysis showed that there were 17 amino acids residues in the N-terminal of the two kinds of sheep SP-A protein,and the subcellular localization analysis showed that the two sheep SP-A proteins were both extracellular.The results of phylogenetic tree analysis showed that Bashibay and Argali hybrid had the closest evolutionary relationship with sheep.(3)The eukaryotic expression vector of SP-A gene of Bashibay and Argali hybrid sheep were confirmed by double enzyme digestion.The results of genotypic and phenotypic identification of Recombinant Pichia pastoris GS115 showed that the SP-A gene of the two sheep was integrated into Pichia pastoris GS115,respectively.The protein expressed in Pichia pastoris weighted26.38 kDa and 26.39 kDa,respectively.(4)The result of plate colony count that when theconcentration of rSP-A was more than 20 ?g/mL,rSP-A could significantly(p<0.05)inhibit the growth of Mo on the Agar plate,indicating that the expressed protein was a bioactive protein.Conclusion:In this study,the full-length cDNA sequence SP-A gene of Bashibay and Argali hybrid sheep was cloned,and the eukaryotic expression,protein purification and detection of protein activity were successfully carried out,which laid a foundation for further study on the structure and function of SP-A genes and SP-A proteins of the two kinds of sheep.