Cloning And Expression And Biological Activity Testing And Effect Of Intramammary Injection Of Rbogm-csf

Cow Mastitis is an inflammation of breast and a complex and the most serious diseases of dairy industry. Although some Western countries has been carried out more than 100 years of this disease research, but there hasn't a way to solve this problem completely.GM-CSF is produced by T cells and macrophages primarily. The main function of GM-CSF is to maintain survival and promote growth, induced differentiation and enhancements of granulocyte cell lines and monocyte cell lines. The purpose of this study is want to find some new methods to therapy cow mastitis by this function of GM-CSF.1. Cloning and expression of Cows GM-CSFBased on the published nucleotide sequence of Bovine GM-CSF(U22385)gene, a pair of RT-PCR primers were designed and synthesized. The 432 bp DNA fragments were amplified by polymerase chain reaction and cloned into pGEM-T vector. The expression plasmid was transformed into BL21 Escherichia coli cell and induced with IPTG . SDS-PAGE confirmed that the expressed protein was about 35kDa . Forecast mature peptide secondary structure and 3D models of cow GM-CSF .The result is that have 4α-helixes and noβ-sheets.2. RboGM-CSF purificationFollowing successfully constructed the pET-32a-GM-CSF expression vector, to adjust the concentration of IPTG and the induced temperature optimize the conditions for inducible expression. SDS-PAGE analysis showed that the majority of recombinant protein is inclusion bodies. After that use urea and Ni-NTA to purify recombinant protein. The results showed that application of urea dissolve inclusion bodies, and then dialysis with decreasing concentration of urea in the buffer to obtain recombinant protein; using Ni-NTA affinity chromatography can obtain a higher purity of the recombinant protein.3. RboGM-CSF biological activity assayUsed Soft agar assay and MTT colorimetric assay detect biological activity of recombinant protein. MTT colorimetric analysis showed that rehabilitation inclusion and Ni-NTA purified protein have activation of maintenance growth and proliferation of lymphocytes.Ni-NTA purification proteins in the concentration of 92μg/ml the strongest capacity to promote lymphocyte proliferation, also showed that is not more higher concentrations of GM-CSF have more capacity to maintain the growth and proliferation of activated lymphocytes.GM-CSF can stimulate the lymphocytes of dairy cows in soft agar, so purification protein with good biological activity.4. Unifies the antibiotic treatment cow subclinical mastitis with rboGM-CSF.New Zealand dairy cows have to choose 48, according to the number of somatic cells into two groups, through the internal mammary single infusion of normal saline, gentamicin, rboGM-CSF and mammary infusion of gentamicin and rboGM-CSF at the same time, to verify the effect of the two group. The results showed that rboGM-CSF alone injection for Mild subclinical mastitis cure rates were 50℅, while 1,000,000/ ml or above did not significantly effect; and gentamicin combination of both groups the cure rates were 83.3℅ and 50℅, rboGM-CSF after injection in addition to short-term temperature rise, there is no side-effects of cows.5. Unifies the traditional Chinese medicine treatment cow mild clinical mastitis with rboGM-CSF.Choice of 16 cows have the initial clinical, milk infusion rboGM-CSF, at the same time feed Ru Yan Bao. The results showed that the cure rate for Ru Yan Bao is 75℅. GM-CSF combined with traditional Chinese medicine, the clinical symptoms disappeared in all cows, the cure rate reached 87.5℅, effective rate is 100℅.