Human S100a1 And S100b Gene Synthesis, Expression, Monoclonal Antibody Preparation And Activity Analysis

Both S100A1 and S100B protein are low molecular weight calcium-binding proteins which belong to S100 protein family. S100 proteins have a wide range of biological activity and functions such as cell proliferation, differentiation, muscle contraction, gene expression, secretion, and apoptosis by affecting calcium signal transduction, affecting hormone secretion, inhibition of tubulin assembly, inhibition of protein kinase C-mediated phosphorylation and others. But the main function is as a calcium receptor-binding protein in the cytoplasm involved in regulating the activity of many cells. Many researches showed that S100 proteins not only express abnormally in many diseases but also be relevant with staging and prognosis of the diseases. Those diseases include psoriasis, Alzheimer disease, cystic fibrosis, cardiomyopathy, muscle atrophy (spinal cord) lateral sclerosis, cancers and so on. The roles of S100 proteins in tumors are not yet clear, but some conclusions suggest that S100 proteins can affect tumor development by regulating the signaling pathways. The members of S100 protein highly expressed in tumor tissues and the antibodies of S100 protein are very significant in the diagnosis and therapy of cancers.Both S100A1 and S100B protein were discovered earliest in all the S100 proteins. The expressions of S100A1 protein are highly tissue-specific and cell specific. They express very little in skeletal muscle but very highly in healthy cardiac cells. S100A1 protein expression decreased in heart failure. The increasing of S100A1 protein expression can improve myocardial contractile and diastolic function, inhibit and reverse the failure in progress by improving the transport of intracellular Ca2+ homeostasis, inhibiting cardiomyocyte apoptosis, improving cardiac remodeling and myocardial energy supplement. S100A1 protein can affect tumor invasion, infiltration and metastasis by the joint with other S100 family members, such as S100A4. In addition, S100A1 protein participates in cell glycometabolism, composition of cytoskeletal dynamics, regulation of cAMP signal transduction pathway associated with G-protein and calcium signal transduction process associated with cell proliferation.S100B protein is the major and most active in S100 protein family members in the brain. About 96% of them exist in the brain and mainly synthesized by astrocytes. S100B protein has a wide range of biological activity and plays an important physiological function under physiological conditions. They have direct neurotoxic effects above normal levels or lead to nerve cells apoptosis interaction with receptor for advanced glycation endproducts (RAGE). S100B protein can regulate cell plasticity, contribute to the calcium handling capacity for astrocytes, participate in information transfer, regulate cell metabolism, nourish the nerves, and so on. In addition, S100B protein can promote islet cell and pituitary tumor cell to secrete insulin and prolactin, and involve in inflammation. S100B protein has the effects of Neurotrophy because them paracrined and autocrined by glial cells can promote neurons and glial cells grow and repair damages.S100A1 and S100B protein are closely associated with the development of many diseases and play their biological roles in the intracellular and extracellular. The researchers have been looking on S100A1 and S100B protein as a marker for the diagnosis of some diseases and potential targets for drug exploration because of the functional diversity of S100A1 and S100B protein. Currently, the researches for the function of S100A1 and S100B protein are comprehensive but little for the mechanisms. What are signaling pathways for S100A1 and S100B proteins in the intracellular and extracellular? Whether do specific receptors exist? What are roles and mechanisms in tumorigenesis? Further researches are necessary. However, we are difficult to get S100A1 and S100B proteins because they mostly depended on import. And the application of proteins and antibodies are limited wildly for their high prices. Therefore, we have been building human protein expression vector for S100A1 and S100B protein and preparing S100A1 and S100B proteins and antibodies of high purity what can be used for clinical detection of tumor tissue and basic researches on corresponding targets in order to reveal the mechanism of cancers; The monoclonal antibodies of S100A1 protein and S100B protein can be used for detection, diagnosis, treatment and prognosis. All of my studies are significant for the further researches. The main results are as follows:Part I: The whole gene synthesis and optimization, expression, purification and identification of human S100A1 and S100B proteinsExperiment I: The whole gene synthesis and optimization, expression, purification and identification of human S100A1 proteinMethods: 1. The human S100A1 gene sequences were synthesized by RT-PCR after optimizated and Ecor I and BamH I restriction sites were introduced on both ends of the primers,2. The cloning vector pMD18-S100A1 was constructed and transformed into E. coli DH5αafter the purified PCR products were inserted into clone vector pMD18-T between the corresponding restriction sites. The plasmid was extracted,purified and digested with Ecor I and BamH I. Then the plasmid was sequenced in Invitrogen Company.3. The expression vector pBV220 and the PCR product were digested by Ecor I and BamH I and linked by T4 DNA ligase. The reconstructed expression plasmid pBV220-S100A1 was transformed into E. coli DH5αand positive transformants were screened by PCR.4. The positive single strain was inoculated in LB medium containing ampicillin and was induced to express S100A1 protein by heat shock.5. The recombinant proteins were charactered by SDS-PAGE and Western Blotting.6. The recombinant proteins were purified by ion exchange chromatography.7. The recombinant proteins were desalted and freeze-dried.Results:1. The S100A1 gene was synthesized and the recombinant expression plasmid BV220-S100A1 was constructed successfully. The results of restriction enzyme digestion and DNA sequencing analysis showed that the S100A1 gene plasmid contained the full coding sequences and the open reading frame was correct.2. The recombinant plasmid PBV220-S100A1 was transformed into expression strain, and the S100A1 protein induced expression was charactered by SDS-PAGE and Western Blotting.Conclusions:1. The recombinant plasmid PBV220-S100A1 including S100A1 coding sequence was constructed successfully.2. We get the purified highly recombinant S100A1 protein after the protein was induced expression by heat shock and purified by ion exchange chromatography. ExperimentⅡ: The whole gene synthesis and optimization, expression, purification and identification of human S100B proteinMethods: 1. The human S100B gene sequences were synthesized by RT-PCR after optimizated and Nde I and XhoⅠrestriction sites were introduced on both ends of the primers,2. The cloning vector pMD18-S100B was constructed and transformed into E. coli DH5αafter the purified PCR products were inserted into clone vector pMD18-T between the corresponding restriction sites. The plasmid was extracted,purified and digested with Nde I and XhoⅠ. Then the plasmid was sequenced in Invitrogen Company.3. The expression vector pET32a and the PCR product were digested by Nde I and XhoⅠand linked by T4 DNA ligase. The reconstructed expression plasmid pET32a-S100B was transformed into E. coli BL21 (DE3) and positive transformants were screened by PCR.4. The positive single strain was inoculated in LB medium containing ampicillin and was induced to express S100B protein by IPTG.5. The recombinant proteins were charactered by SDS-PAGE and Western Blotting.6. The recombinant proteins were purified by ion exchange chromatography.7. The recombinant proteins were desalted and freeze-dried.Results:1. The S100B gene was synthesized by whole gene synthesis and the recombinant expression plasmid pET32a-S100B was constructed successfully. The results of restriction enzyme digestion and DNA sequencing analysis showed that the S100B gene plasmid contained the full coding sequence and the open reading frame was correct.2. The recombinant plasmid pET32a-S100B was transformed into expression strain, and the protein induced expression is S100B charactered by SDS-PAGE and Western Blotting.Conclusions:1. The recombinant plasmid pET32a-S100B including S100B coding sequence was constructed successfully.2. We get the purified highly recombinant S100B protein after the protein was induced expression by IPTG and purified by ion exchange chromatography.Part II: The study of Human S100A1 and S100B protein in promoting Hela cell invasion and migration.Methods: The matrix membranes were analoged in vivo with artificial basement membrane and Matrigel. The ability of invasion for tumor cells were reflected indirectly according to the number of cells throughed the matrix membrane. And the number of tumor cells throughed the membrane without Matrigel reflected indirectly its ability of migration. The membrane covered with martrigel was between two chambers and martrigel was in upper chamber. The chemotactic agents were in lower chambers. The mixture of 100ul Hela cells suspension and 100ul basal medium were added to the upper rooms for the control group, otherwise, 100ul cells suspension and 100ul recombinant protein were added for the experimental group. The cells with the ability of invasion may move towards the chemotactic agents. Most of the cells through the membrane would adhere to surface of the membrane. Then the cells in the upper surface were wiped with a cotton swab. The number of cells through martrigel may be statisticed after cells were fixed and stainedResults:1. Human S100A1 protein can significantly improve the migration ability of Hela cell (P <0.05), but had no significant effect on the invasion ability of Hela cells.2. Human S100B protein increased the invasion ability of Hela cells to 2.8 times, and the migration ability to 3.5 times, which means S100B protein can promote Hela cell invasion and migration ability.Conclusions:1. The purified S100A1 proteins had biological activity and could improve tumor cell migration ability. However, there was less effect on the invasive ability of tumor cells.2. The purified S100B proteins had biological activity and could improve the invasion and migration ability of tumor cell. Part III: A monoclonal antibody against human S100A1 and S100B preparation, purification and identification Experiment I: A monoclonal antibody against human S100A1 preparation, purification and identificationMethods:1. The purified recombinant S100A1 protein and immune adjuvant were mixed and immunized the mice.2. The immuned spleen cells were fused with mouse myeloma cells with conventional cell fusion method. 3. The titer of against S100A1 in the hybridoma cells supernatant were detected by ELISA and positive clones were screened.4. The single cell strain which could secrete antibody against S100A1 was gotten after the positive cell strains were subcloned by limited dilution.5. The stability that the single cell strain secreted antibody was detected by which positive cell lines were cryopreserved and recovered after three months.6. The hybridoma single cell strains were vaccinated into mices and ascitic fluids were extracted. The monoclonal antibody against S100A1 was purified with Protein G HP.7. The antibody specificity was identified by Western Blotting and ELSIA.8. The affinity constants of antibodies were identified by ELSIA.Results:1. Four cell lines which could secret anti-human S100A1 antibodies were obtained.2. The affinity constant of four antibodies were very high which meaned antigen binding capacity were good, and the four antibodies have no cross reactions with other antigens.Conclusions:1. The hybridoma cell lines which could secret anti-human S100A1 antibody stably were established.2. The monoclonal antibody against human S100A1 were prepared and identified.ExperimentⅡ: A monoclonal antibody against human S100B preparation, purification and identificationMethods:1. The purified recombinant S100B protein and immune adjuvant were mixed and immunized the mice.2. The immuned spleen cells were fused with mouse myeloma cells with conventional cell fusion method.3. The titer of against S100B in the hybridoma cells supernatant were detected by ELISA and positive clones were screened.4. The single cell strain which could secrete antibody against S100B was gotten after the positive cell strains were subcloned by limited dilution.5. The stability that the single cell strain secreted antibody was detected by which positive cell lines were cryopreserved and recovered after three months. 6. The hybridoma single cell strains were vaccinated into mices and ascitic fluids were extracted. The monoclonal antibody against S100B was purified with Protein G HP.7. The antibody specificity was identified by Western Blotting and ELSIA.8. The affinity constants of antibodies were identified by ELSIA.Results:1. Three cell lines which could secret anti-human S100B antibodies were obtained.2. The affinity constant of three antibodies were very high which meaned antigen binding capacity were good, and the three antibodies had no cross reactions with other antigens.Conclusions:1. The hybridoma cell lines which could secret anti-human S100B antibody stably were established.2. The monoclonal antibody against human S100B were prepared and identified. Part IV: The activity analysis of the monoclonal antibodies against human S100A1 and S100B.Methods:1 Cell proliferation, apoptosis and P53 protein expression were observed after exogenous monoclonal antibody against human S100A1 protein were added into the human melanoma A375.2 The expression of S100A1 protein in A375 human melanoma cells were charactered with the prepared monoclonal antibody against human S100A1 protein and commercial monoclonal antibody against human S100A1 protein.3 Cell proliferation, apoptosis and P53 protein expression were observed after exogenous monoclonal antibody against human 100B protein were added into the human melanoma A375.4 The expression of S100B protein in A375 human melanoma cells were charactered with the prepared monoclonal antibody against human S100B protein and commercial monoclonal antibody against human S100B protein.Results:1 The exogenous monoclonal antibody against human S100A1 could slow cell proliferation, increase the cell apoptosis and P53 protein expression,decrease S100A1 protein expression.2 Western Blotting analysis showed that the expression of S100A1 protein in the A375 human melanoma cells can be detected weakly and the detection results were basically same with the commercial monoclonal antibody.3 The exogenous monoclonal antibody against human S100B could slow cell proliferation, increase the cell apoptosis and P53 protein expression,decrease S100B protein expression.4 Western Blotting analysis showed that the expression of S100B protein in the A375 human melanoma cells can be detected and the detection results were basically same with the commercial monoclonal antibody.Conclusions:1. The monoclonal antibody against human S100A1 protein and S100B protein can be used for Western Blotting detection and can inhibit tumor cell proliferation, which can play the role of targeted therapy for a variety of cancer diagnosis and therapy.2. The monoclonal antibody against human S100A1 and S100B can increase the expression of wild-type P53 protein in cancer cells, which can promote apoptosis and inhibit tumor cell proliferation.