Study On Purification, Purity Analysis Of Haptoglobin And Its Relevancy With Cow Matitis

Haptoglobin(Hp),one of acute phase proteins,is an acid glycoprotein.Hp polymorphism arises from variantα-chains.Synthesis of Hp is mostly in liver, Haptoglobin expression is induced by lipopolysaccharide,cytokine and hormone. Haptoglobin polymorphism is associated with the prevalence and clinical evolution of many inflammatory diseases,including infections,atherosclerosis,and autoimmune disorders.Mastitis is one of cows' common disease which has another name called mamma inflammation.Pathogenic microorganism(infection pathogenic microorganism and environmental agents) invading the mamma.And other factors(other diseases continue, improperly raising management) can cause the mastitis,decreases the defence capabilities which makes the galactophore inflammation and changes the component of milk.Mastitis makes the mamma tissue demolished,influences the function of mamma,reduces milk yield,and causes great waste of fodder,medicine and workforce.The nutritive value of the rotten milk drops notably at the same time,not only endanger people's health,but alsol bring the enormous economic losses to the cow industry.The researches of haptoglobin have already been relatively deepened at present.But the research of cow haptoglobin have just begun,mastitis has not been confirmed and verified have relation with haptoglobin yet.The purified condition of separation of haptoglobin in bovine plasma and qualification method also remain to be clarified.This text regards cows as the research object,has made and compared haptoglobin with the blood of the healthy cows and cows who suffered from mastitis tentatively;abstraction plasma,separating purifying and purification determine that has carried on the research of haptpglobin in serum;study the kinds of inferior base of the cow haptoglobin;analyze the relevancy that the change of haptoglobin content in bovine plasma and cow's mastitis. Probe into the relevancy of serum haptoglobin with farrowing of cow count,breeding ability and milk yield,selecting better hereditary properties,it is the performance of cultivating,improving hereditary structure to offer the reliable theoretical foundation. Coagulating Cow blood to make serum with the centrifuge,analyse the differences of protein between healthy cow serum and cow plasma which suffering from mastitis with the SDS-PAGE,thus obtaining haptoglobin in cow plasma which is suffered from mastitis,while there is no haptoglobin in the healthy ones,which can confirm that haptoglobin and mastitis that have relevancy.Serum of the mastitis cow was extracted by PBS buffer solution and NaCl solution at 4℃overnight.Then deposited by(NH₄)₂SO₄.According to the effect factor of PBS pH, salt concentration and extracted ratio,a 23 factorial design of orthogonal rotation was performed to find the best conditions of extraction.The best conditions were:PBS pH, salt concentration,extracted ratio.The best obtain percent was:PBS pH6.8,salt concentration0.15 mol/l,extracted ratio 1:10The deposition of the cow serum was dissolved in distilled water,dialyzed with PBS buffer solution and concentrated by PEG-6000,then separated with DEAE-Cellulose 52 IEC.After being eluted by PBS buffer solution,PBS-0.1mol/l NaCl and PBS-0.2mol/l NaCl,the serum protein was collected.At the second place,the serum protein was purified on Sephacryl S-300 SEC,and was collected.SDS-PAGE and HPLC were used to analyse the purification of haptoglobin,verify it is the pure product.Haptoglobin can be degraded for the inferior base with the cysteine and 2-mercaptoethanol,after the demotion,SDS-PAGE can be used to analyze its purity.