The Research Of Separating, Purifying, Identifying Marker Proteins From Primary Hepatic Carcinoma Serum And Corresponding Antibody Preparation

Objective: 1. To explore and ascertain the best means of separating and purifying micromolecular proteins through using proteomics technique: HPLC,CE,SDS-PAGE,MS,et al to separate, purify and identify 9 worthying of diagnosing markers proteins which had been screend by SELDI before our research. 2.To identify the property of the separated and purified marker proteins by mass spectrum identification and bioinformatics analysis. 3.To prepare the polyclonal antibodies of the separated and purified marker proteins for their further research.Methods:1.Serum-albumin removal:Used respectively isoelectric precipitation, ammonium sulfate precipitation,Hitrap Blue affinity chromatography,and ultrafiltration to remove serum-albumin from serum and employed electrophoresis,SELDI technique to detect protein concentration,surveyed the status of serum-albumin removal and aim protein residue, and then ascertained the best empirical method of serum-albumin removal.2.Tricine-SDS-PAGE electrophoretic separation and gel protein reclamation: Separated and purified preliminarily aim proteins from the samples from which albumin was removed by Tricine-SDS-PAGE electrophoretic method,identified the reclamation proteins by SELDI.3. Tandem mass Spectrometry was used to separate and identiy the aim proteins further,and bioinformatics was employed to analysed aim proteins. 3.1 HPLC-MS separation and identifition and bioinformatics analysis:Cuted and digested the gel strip of the definitive marker proteins,identified the aim proteins by HPLC-MS,and then analysed the results by bioinformatics.3.2 CE-MS separation and identification: Founded CE-MS protein identification methods through nominal sample BSA,and then separated and identified the samples containing the aim proteins by the CE-MS proteins method.4.Polyclonal antibody preparation: Prepared the polyclonal antibody of the marker proteins which had been separated, purified,and identified.Results:1.After the abundant serum-albumin were removed from the serum samplesthe respectively by isoelectric precipitation, ammonium sulfate precipitation,Hitrap Blue affinity chromatography and ultrafiltration,and the contents of aim proteins were detected by electrophoresis and CM-10 protein chip,the results showed that PH5.6 isoelectric precipitation removed the most serum-albumin and made the most aim proteins remained,the strip of aim protein was clear in gel,so PH5.6 isoelectric precipitation was considered preliminarily as the first step of separating and purifying marker proteins from primary hepatic carcinoma serum;ammonium sulfate precipitation and ultrafiltration were not effective on albumen removal; though Hitrap Blue affinity chromatography removed the albumen effectively,no stained strips appeared below 10KD range in running gel.2.After the primary hepatic carcinoma serum(or normal contral serum) was treated by PH5.6 isoelectric precipitation first,and then Tricine-SDS-PAGE electrophoresis,the gel results showed that clear protein strips appeared in the range of the aim proteins'molecular weight(6.5—9.5KD and 9.5KD-14.4KD respectively)3.Cutted the aim protein strip first,then collected electroeluate after electroeluting,next detected the aim proteins by CM-10 protein chip.The results showed that the method was effective to recover the aim proteins 8710Da,8596Da,and nontarget protein was significantly removed as well.4.The aim protein(8710Da,8596Da) strip was conducted by LC-ESI-MS treatment and software Mascot analysis.The results showed that the aim protein was identified as Apo AII ,Mowse Score was 79(P<0.05).5.The results from bioinformatics analysis showed that the protein (P02652) was prepro-Apo AII,an immature secretary protein,in which an amino acid chain contains 77(or 76) amino acids ,MW=8 707.9Da (or MW=8579.78), pI=5.05,MW was equal to that of the aim proteins 8710.19Da,8579.07Da (pI4.0-5.6).6.The zymolyte of nominal sample BSA was identified as BSA by CE-MS identification and software Macot retrieval,Mowse Score was 210.However, CE-MS was employed to separate and identify the inspissated electroeluate containing aim proteins,no credible proteins were found out.The empirical method should be improved further.7.The polyclonal antibody of aim protein 7984M/Z was prepared,antibody titer was 1:729000,the OD curve of immune serum was higher than that of preimmune serum,its crossover experiments with other two antibodies were both negative.The results of purified antibody electrophoresis suggested that only 50KD and 25KD protein strips were appeared which was consistent with MW of IgG's H and L strand.Conclosion:1.PH5.6 isoelectric precipitation which could not only made the most aim protein remained but also removed the abundant nontarget proteian most effectively was ascertained as the first step of separating and purifying preliminarily marker proteins from primary hepatic carcinoma serum.2.Tricine-SDS-PAGE electrophoresis which was proved a convenient and effective means of micromolecular proteins separation was ascertained as the second step of separating and purifying further marker proteins from primary hepatic carcinoma serum.3.The third step:the aim proteins which was separated and purified by electrophresis could be separated and identified further by two methods.One was HPLC-MS/MS technique,that separeted and identified the aim proteins 8710Da and 8596 Da successfully suggested it could separate and identify partial aim proteins effectively.The other was CE-MS/MS technique,though using CE-MS/MS to separate and identify the aim protein did not attain satisfying results,the method could separate and identify the zymolyte of nominal sample BSA suggested that CE-MS/MS was still a worthy method to separate and identify aim proteins.4.The fourth step was using the purified marker proteins from primary hepatic carcinoma serum to prepare polyclonal antibody.That the polycloal antibody of marker protein 7984M/Z could be prepared indicated that a series of technique system from screening marker protein,separating,purifying,identifying to preparing corresponding antibody had been built preliminarily.