Cloning And Sequence Analysis Of Goat GM-CSF Gene And LppA Gene

Granulocyte-macrophage colony stimulating factor(GM-CSF)is made by a variety of cells including the activated T cell,B cell,Macrophage,Mast cells,Endothelial cell,Fibrocyte in response to antigenic and cell factor induction.GM-CSF can add the quantity of Dendritic cell and reinforce the Dendritic cell's capability of antigen presentation,that played a very important role in immune response which induced by DNA vaccination.It can also add the quantity of IL-2,activate CD4~+T cell and enhance the ability of secretory antibody.It simultaneously can increase the biological activity of CD8~+T cell.So it can be used as immunoadjuvant of DNA vaccine and add its immunogenicity.Infectious pleuropneumonia of goats is a disease which make goats culturist feel troublesome.At clinic,diseased goats are always fever heat,coughing and thin.And the other diseased goats of prolonged illness often becomes loses procreative instinct.The direct losses are high mortality,the dropped rate of producing milk or meat and that the cost of the disease control,diagnose,treat is too high.So,it is one of serious goat's diseases which the mainly feeding goats countries have to face in the world.M.mycoides subsp.capri is the primary pathogen of infectious pleuropneumonia of goats. M.mycoides subsp.capri is tiny and pantomorphic microorganism.The lipoprotein of LppA is the significant antigenic composition.It is seldom report of the protein secondary structure,hydrophilism area and antigen index.First of all,extracted the total RNA from goat splenic lymphocyte and based on the published nucleotide sequence of goat GM-CSF,a pair of primers was designed and synthesized.With the technology of RT-PCR,a cDNA fragment was amplified,and cloned to pMD18-T Vector.cDNAs encoding goat GM-CSF were cloned and sequence analysis showed that they were 435 bp in length and encoded a predicted mature protein of 144 amino acids.Compared with ovine, bovine,porcine,equus,baboon,macaca mulatta,human,canine,felis,murine and fowl GM-CSF, homology of the goat GM-CSF coding sequence was,respectively,97.7%,91.5%,86.5%,88.3%,83.7%, 84.9%,85.3%,83.0%,84.9%,73.1%and 39.1%at the nucleotide level.Experimental result showed that goat GM-CSF gene was cloned.Secondly,according to the sequence of lppA gene in Mycoplasma mycoides subsp.capri.PG3 strain,pairs of primers were designed to amplify lppA gene.Used of DNAStar to analyze the secondary sturcture of protein,hydrophilism district and index of antigen of the lppA gene.The sequence analysis showed that they were 1256 bp in length.The nucleotide sequence of the lppA gene contained an open reading frame(ORF)encoding the lipoprotein LppA precursor of 523 amino acid residues.DNA sequence and amino acid sequence of LppA were compared with sequences reported and found that the base changed at the seat of 67,136 and 676,the 20 and 223 amino acid changed and another amino acid had samesense mutation.Lipoprotein LppA had 15α-coiling areas, 19β-folding areas and many inflection points.LppA for the most part was hydrophilic and had high antigenicity.The analysis of rare codon in LppA indicated that LppA had two codons UGA which coded Trp,in Bacillus coli UGA was stop codon,that induced lppA gene had no expression in Bacillus coli.Subsequent experiment should considered to change TGA into TGG by genetic mutation.In conclusion,cDNA encoding goat GM-CSF and LppA gene was successfully cloned and sequencing.GM-CSF gene possesses species specificity.Used of DNAStar to analyze the secondary sturcture of protein,hydrophilism district and index of antigen of goat GM-CSF gene and lppA gene.