Separation And Quantitative Analysis Of Medicinal Surfactant Protein

Pulmonary surfactant protein(SP)is secreted from type ? alveolar cells,and it is specific binding with pulmonary surfactant(PS).SP has two types,one kind of small molecular hydrophobicity protein SP-B and SP-C,the other kind of macromolecular hydrophilic protein SP-A and SP-D.Among them,the hydrophobicity proteins are significant for the activity of pulmonary surfactant.At present,there have been related products used in clinical treatment of acute lung injury/acute respiratory distress syndrome and other diseases.But the amount of target protein is uncertain,it needs to prepare the standard substance to analysis and establish the corresponding test methods.Our resarch is to establish a stable method of preparation of surfactant protein SP-B and SP-C.First of all,we purify SP-B and SP-C from the raw material of cattle lung rinses with the aids of frozen processing of organic solvent,solid phase extraction method.To optimize the conditions of the extraction and separation,including extraction solvents,extraction times,etc.The results show that,Using ethyl acetate/chloroform dissolve for cold treatment,centrifugal,repeat extraction 5 times,it can remove 65.7% impurities with 8.7% of the total protein lost,and than for solid phase extraction processing,and it can remove 83.3%impurities with 26.5% of the total protein lost.Second,using the high performance liquid chromatography(HPLC)and enzymelinked immunosorbent(ELISA)to analyze purity of samples before and after purification.High performance liquid chromatography(HPLC)analysis showed,For each methods of the purification,the chromatograph chart after purification compared to before purification,the number of eluting peak reduced,which illustrated impurities were reduced.Enzyme-linked immunosorbent experimental results display,first with organic solvent cold treatment and than solid phase extraction processing,whose effect of purification is best.After this operation,it can remove 83.3%impurities,and the content of SP-B ascended from the original 0.612 ?g/mg to 3.075 ?g/mg,and its yield of the target protein is 84.268%,the content of SP-C also got up from 0.324 ?g/mg to 1.706 ?g/mg,and its yield of the target protein is 87.865%.