人源S100A1和S100B全基因合成、表达、单抗制备及其活性分析
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摘要
S100A1和S100B蛋白是低分子量钙结合蛋白,均属于S100蛋白家族。S100蛋白家族分子具有广泛的生物学活性。其主要功能是作为钙结合蛋白参与多种细胞内的活性调节,通过钙离子信号转导、影响激素分泌、抑制微管蛋白的组装及抑制蛋白激酶C介导的磷酸化等途径,在细胞增殖与分化、肌肉收缩、基因表达与分泌、以及细胞凋亡中发挥重要作用。研究表明,S100基因或蛋白在很多疾病中表达异常,如阿希海默病、银屑病、胆囊纤维化、肌萎缩性(脊髓)侧索硬化、心肌病和癌症等,而且与疾病的分期和预后密切相关。目前,S100蛋白家族在肿瘤发生发展中的作用尚不明确。但已有研究提示,S100蛋白家族通过调节信号通路影响肿瘤发生发展。S100蛋白家族成员在多种肿瘤组织中高表达,其抗体在肿瘤的诊断和治疗中具有很重要的潜在作用和意义。
S100A1和S100B蛋白是最早被发现的S100蛋白家族成员。S100A1蛋白的表达有高度的组织特异性和细胞特异性,在骨骼肌中很少表达,而在健康心肌细胞中则大量表达。心力衰竭时S100A1表达下降,增加S100A1的表达能改善心肌细胞内Ca2+转运的动态平衡,抑制心肌细胞凋亡,改善心肌能量供应,影响心脏重构,从而改善心肌收缩、舒张功能,抑制和逆转心力衰竭的进展。S100A1可与其它S100家族成员如S100A4共同作用,影响肿瘤的浸润、侵袭和转移。此外,它还参与细胞糖代谢,与细胞骨架动力学组成相关,通过与G蛋白作用调控cAMP的信号传导途径,参与细胞增殖相关的钙信号传导过程。
S100B是S100家族中存在于脑内最主要和最具有活性的成员,约96%存在于脑内,主要由星形胶质细胞合成。S100B蛋白有广泛的生物学活性,在生理情况下发挥重要的生理功能,但含量过高可能具有直接神经毒性作用,或者同糖基化终末产物的受体(receptor for advanced glycation endproducts,RAGE)相互作用而导致神经细胞凋亡。S100B能调节细胞的可塑性,有助于星形胶质细胞的钙处理能力,参与信息传递,调节细胞代谢,有胶质源性的营养作用。此外, S100B能促进胰岛细胞、垂体瘤细胞分泌胰岛素和催乳素,参与炎症反应。神经胶质细胞旁分泌和自分泌S100B蛋白,可作用于神经元和神经胶质细胞,促进神经的生长和损伤的修复。
S100A1和S100B蛋白与很多疾病的发生发展有密切联系,在胞内和胞外均能发挥其生物学作用。由于S100A1和S100B蛋白功能的多样性,研究人员把S100A1和S100B蛋白作为某些疾病诊断的标志物以及潜在药物靶标进行探索,并已取得了阶段性研究成果。目前,对S100A1和S100B蛋白的功能研究比较全面,但开展S100A1和S100B蛋白作用机制方面的研究很少,S100A1和S100B蛋白在细胞内外发挥作用的信号转导途径是什么?是否存在特异性受体?其在肿瘤发生发展中的作用及机制如何?需要进一步研究确定。但S100A1和S100B蛋白及其单克隆抗体多为进口产品,价格高,购买困难,无论从经济上还是效率上都限制了对该蛋白和抗体的研究和利用,不利于大规模地开展蛋白相关功能及其机制的研究。因此,我们拟构建人源S100A1和S100B蛋白表达载体,制备高纯度国产人源S100A1和S100B蛋白及其单克隆抗体。制备获得的重组蛋白,可用于肿瘤组织的临床检测及分子靶标作用的基础研究,以揭示其作用机制。制备其单克隆抗体可用于人源S100A1和人源S100B蛋白表达水平的检测、疾病诊断、治疗及预后判断,这为以后深入研究该蛋白特性及其在相关疾病中的作用机制奠定了基础,具有重要的现实意义。主要研究结果如下:第一部分:人源S100A1和S100B全基因合成和优化、表达、纯化与鉴定
实验一人源S100A1全基因合成和优化、表达、纯化与鉴定
方法:
1.优化人源S100A1基因序列,设计合成人源S100A1序列引物,在两端引物分别引入EcoR I和BamH I酶切位点,分段合成法PCR全基因合成人源S100A1全基因。
2.回收纯化PCR产物, TA克隆至载体pMD18-T ,构建克隆载体pMD18-S100A1,并将其转化到E. coli DH5α中,PCR筛选阳性转化子,小量提取质粒,经酶切鉴定后送invitrogen公司测序。
3.用EcoR I和BamH I双酶切测序正确的pMD18-S100A1,与经同样酶切的pBV220质粒用T4 DNA连接酶连接,构建表达质粒pBV220 -S100A1,并将其转化到表达宿主E.coli DH5α中,PCR筛选阳性转化子。
4.将阳性单菌接种于含氨苄西林的LB培养基中,热激诱导表达人源S100A1蛋白。
5. SDS-PAGE和Western Blotting分析鉴定重组蛋白。
6.采用离子交换层析法纯化人源S100A1蛋白。
7.脱盐并冻干人源S100A1蛋白。
结果:
1.全基因合成了人源S100A1基因,成功构建重组表达质粒pBV220-S100A1,经酶切鉴定和DNA序列测序分析表明,该质粒含有人源S100A1基因的编码序列全长,开放阅读框架正确。
2.重组表达质粒pBV220-S100A1转化入表达宿主,诱导表达产物经纯化、SDS-PAGE和Western Blotting分析,表明表达蛋白为人源S100A1蛋白。
结论:
1.成功构建含有人源S100A1编码序列原核表达载体pBV220-S100A1。
2.经热激诱导表达、离子交换层析纯化获得纯度达95%的重组S100A1蛋白。
实验二人源S100B全基因合成和优化、表达、纯化与鉴定
方法:
1.优化人源S100B基因序列,设计合成人源S100B序列引物,在两端引物分别引入Nde I和XhoⅠ的酶切位点,分段合成法PCR合成人源S100B基因。
2.回收纯化PCR产物,TA克隆至载体pMD18-T,构建克隆载体pMD18-S100B,并将其转化到E. coli DH5α中,PCR筛选阳性转化子,小量提取质粒,经酶切鉴定后送invitrogen公司测序。
3.用Nde I和XhoⅠ双酶切测序正确的pMD18-S100B,与经同样酶切的pET32a质粒用T4 DNA连接酶连接,构建表达质粒pET32a-S100B,并将其转化到表达宿主E.coli BL21(DE3)中,PCR筛选阳性转化子。
4.将阳性单菌接种于含氨苄西林的LB培养基中,IPTG诱导表达人源S100B蛋白。
5. SDS-PAGE和Western Blotting分析鉴定重组蛋白。
6.采用离子交换层析法纯化人源S100B蛋白。
7.脱盐并冻干人源S100B蛋白。
结果:
1.全基因合成人源S100B基因,成功构建重组表达质粒pET32a-S100B,经酶切鉴定和DNA序列测序分析表明,该质粒含有人源S100B基因的编码序列全长,开放阅读框架正确。
2.重组表达质粒pET32a-S100B转化入表达宿主,诱导表达产物经纯化、SDS-PAGE和Western Blotting分析,表明表达蛋白为人源S100B蛋白。
结论:
1.成功构建含有人源S100B编码序列原核表达载体pET32a-S100B。
2.经IPTG诱导表达、离子交换层析纯化获得纯度≥95%的重组S100B蛋白。
第二部分:人源S100A1和S100B蛋白促进Hela细胞侵袭迁移研究
方法:
利用人工基底膜和基质胶,模拟在体的基质膜屏障,根据肿瘤细胞穿过基质膜的数量间接反映它的侵袭能力,而肿瘤细胞穿过不含基质胶滤膜的数量间接反映它的迁移能力。铺有基质胶的滤膜放在上下室之间,铺有基质胶面朝向上室,在下室中加趋化剂,对照组上室加入100uL重悬的Hela细胞和100uL基础培养基,实验组上室加入100uL重悬的Hela细胞和100uL重组蛋白,具有侵袭能力的细胞在趋化剂诱导下开始穿膜运动。穿过滤膜的细胞多数粘附在滤膜下表面,可用棉签将上表面的细胞拭去,然后经固定、染色,观察统计穿过基质胶的细胞数量。
结果:
1.人源S100A1蛋白显著提高Hela细胞迁移能力(与对照组相比,P<0.05),但对其侵袭能力无显著影响。
2.人源S100B蛋白可使Hela细胞的侵袭能力提高2.8倍,迁移能力提高3.5倍。
结论:
1.纯化获得的重组人源S100A1蛋白具有生物活性,可提高肿瘤细胞的迁移能力。但对其侵袭能力影响较小。
2.纯化获得的重组人源S100B蛋白具有生物活性,可提高肿瘤细胞的侵袭和迁移能力。
第三部分:抗人S100A1和S100B蛋白单克隆抗体的制备、纯化与鉴定
实验一抗人S100A1蛋白单克隆抗体的制备、纯化与鉴定
方法:
1.将纯化的重组人源S100A1蛋白和免疫佐剂共同制备成免疫原,免疫小鼠。
2.用传统的细胞融合法,将免疫成功的小鼠脾细胞与骨髓瘤细胞融合得到杂交瘤细胞。
3. ELISA检测杂交瘤细胞上清液中抗人S100A1抗体的效价,筛选阳性细胞株。
4.有限稀释法对阳性细胞株进行亚克隆,得到能够分泌目标抗体的单细胞株。
5.冻存阳性细胞株,三个月后复苏细胞,检测其抗体分泌的稳定性。
6.体内接种杂交瘤单细胞株,制备腹水型单克隆抗体,用Protein G HP层析纯化蛋白,得到人源S100A1单克隆抗体。
7. Western Blotting及ELSIA法鉴定抗体特异性。
8. ELSIA法测定抗体的亲和常数。
结果:
1.获得了4株能稳定分泌抗人S100A1单克隆抗体细胞株。
2. 4株细胞抗体亲和常数均较高,结合抗原的能力均较强,与其他抗原无交叉反应,特异性好。
结论:
1.建立了能稳定分泌抗人S100A1单克隆抗体的杂交瘤细胞株。
2.制备出抗人S100A1单克隆抗体,并进行了相关性质鉴定。
实验二抗人S100B蛋白单克隆抗体制备、纯化与鉴定
方法:
1.将纯化的重组人源S100B蛋白和免疫佐剂共同制备成免疫原,免疫小鼠。
2.用传统的细胞融合法,将免疫成功的小鼠脾细胞与骨髓瘤细胞融合得到杂交瘤细胞。
3. ELISA检测杂交瘤细胞上清液中人源S100B抗体的效价,筛选阳性细胞株。
4.有限稀释法对阳性细胞株进行亚克隆,获得能分泌目标抗体的单细胞株。
5.冻存阳性细胞株,三个月后复苏细胞,检测其抗体分泌的稳定性。
6.体内接种杂交瘤单细胞株,制备腹水型单克隆抗体,用Protein G HP纯化蛋白,获得人源S100B单克隆抗体。
7. Western Blotting及ELSIA法鉴定抗体特异性。
8. ELSIA法测定抗体的亲和常数。
结果:
1.获得了3株能稳定分泌抗人S100B单克隆抗体的细胞株。
2. 3株细胞抗体亲和常数均较高,结合抗原的能力均较强,与其它抗原无交叉反应,特异性好。
结论:
1.建立了能稳定分泌抗人S100B单克隆抗体的杂交瘤细胞株。
2.制备出抗人S100B单克隆抗体,并进行了相关特性鉴定。
第四部分:抗人S100A1和S100B单克隆抗体活性分析
方法:
1.外源性给予抗人S100A1单克隆抗体,观察其对人黑色素瘤细胞A375增殖、凋亡及P53蛋白表达的影响。
2.以商业化的抗人S100A1单克隆抗体为对照,利用制备的抗人S100A1单克隆抗体Western Blotting法检测人黑色素瘤细胞A375中S100A1蛋白的表达。
3.外源性给予抗人S100B单克隆抗体,观察其对人黑色素瘤细胞A375增殖、凋亡及P53蛋白表达的影响。
4.商业化的抗人S100B单克隆抗体为对照,利用制备的抗人S100B单克隆抗体Western Blotting法检测人黑色素瘤细胞A375中S100B蛋白的表达。
结果:
1.外源性给予抗人S100A1单克隆抗体,细胞增殖速度减慢,细胞凋亡增加,胞浆内S100A1蛋白表达降低,P53蛋白表达增多。
2. Western Blotting检测显示,S100A1蛋白在人黑色素瘤细胞A375中弱表达,且与商业化的进口单抗相比,检测效果无明显差异。
3.外源性给予抗人S100B单克隆抗体,细胞增殖速度减慢,细胞凋亡增加,胞浆内S100B蛋白表达降低,P53蛋白表达增多。
4. Western Blotting检测显示,S100B蛋白在人黑色素瘤细胞A375中有表达,但与商业化的进口单抗相比,检测效果无明显差异。
结论:
1.制备的抗人S100A1和S100B单克隆抗体既可以用于肿瘤细胞的Western Blotting检测,又可发挥抑制肿瘤细胞的增殖,起到靶向治疗作用,为相关疾病的诊断和治疗提供了新的筛查手段。
2.抗人S100A1和S100B单克隆抗体对癌细胞的靶向治疗作用,其机制可能是通过与S100A1和S100B蛋白结合,使胞浆内S100A1和S100B蛋白表达减少,增加了野生型P53蛋白表达,从而促细胞凋亡,达到抑制肿瘤细胞增殖的作用。
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 proteins
Experiment I: The whole gene synthesis and optimization, expression, purification and identification of human S100A1 protein
Methods:
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 protein
Methods:
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 stained
Results:
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 identification
Methods:
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 identification
Methods:
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.
S100A1和S100B蛋白是最早被发现的S100蛋白家族成员。S100A1蛋白的表达有高度的组织特异性和细胞特异性,在骨骼肌中很少表达,而在健康心肌细胞中则大量表达。心力衰竭时S100A1表达下降,增加S100A1的表达能改善心肌细胞内Ca2+转运的动态平衡,抑制心肌细胞凋亡,改善心肌能量供应,影响心脏重构,从而改善心肌收缩、舒张功能,抑制和逆转心力衰竭的进展。S100A1可与其它S100家族成员如S100A4共同作用,影响肿瘤的浸润、侵袭和转移。此外,它还参与细胞糖代谢,与细胞骨架动力学组成相关,通过与G蛋白作用调控cAMP的信号传导途径,参与细胞增殖相关的钙信号传导过程。
S100B是S100家族中存在于脑内最主要和最具有活性的成员,约96%存在于脑内,主要由星形胶质细胞合成。S100B蛋白有广泛的生物学活性,在生理情况下发挥重要的生理功能,但含量过高可能具有直接神经毒性作用,或者同糖基化终末产物的受体(receptor for advanced glycation endproducts,RAGE)相互作用而导致神经细胞凋亡。S100B能调节细胞的可塑性,有助于星形胶质细胞的钙处理能力,参与信息传递,调节细胞代谢,有胶质源性的营养作用。此外, S100B能促进胰岛细胞、垂体瘤细胞分泌胰岛素和催乳素,参与炎症反应。神经胶质细胞旁分泌和自分泌S100B蛋白,可作用于神经元和神经胶质细胞,促进神经的生长和损伤的修复。
S100A1和S100B蛋白与很多疾病的发生发展有密切联系,在胞内和胞外均能发挥其生物学作用。由于S100A1和S100B蛋白功能的多样性,研究人员把S100A1和S100B蛋白作为某些疾病诊断的标志物以及潜在药物靶标进行探索,并已取得了阶段性研究成果。目前,对S100A1和S100B蛋白的功能研究比较全面,但开展S100A1和S100B蛋白作用机制方面的研究很少,S100A1和S100B蛋白在细胞内外发挥作用的信号转导途径是什么?是否存在特异性受体?其在肿瘤发生发展中的作用及机制如何?需要进一步研究确定。但S100A1和S100B蛋白及其单克隆抗体多为进口产品,价格高,购买困难,无论从经济上还是效率上都限制了对该蛋白和抗体的研究和利用,不利于大规模地开展蛋白相关功能及其机制的研究。因此,我们拟构建人源S100A1和S100B蛋白表达载体,制备高纯度国产人源S100A1和S100B蛋白及其单克隆抗体。制备获得的重组蛋白,可用于肿瘤组织的临床检测及分子靶标作用的基础研究,以揭示其作用机制。制备其单克隆抗体可用于人源S100A1和人源S100B蛋白表达水平的检测、疾病诊断、治疗及预后判断,这为以后深入研究该蛋白特性及其在相关疾病中的作用机制奠定了基础,具有重要的现实意义。主要研究结果如下:第一部分:人源S100A1和S100B全基因合成和优化、表达、纯化与鉴定
实验一人源S100A1全基因合成和优化、表达、纯化与鉴定
方法:
1.优化人源S100A1基因序列,设计合成人源S100A1序列引物,在两端引物分别引入EcoR I和BamH I酶切位点,分段合成法PCR全基因合成人源S100A1全基因。
2.回收纯化PCR产物, TA克隆至载体pMD18-T ,构建克隆载体pMD18-S100A1,并将其转化到E. coli DH5α中,PCR筛选阳性转化子,小量提取质粒,经酶切鉴定后送invitrogen公司测序。
3.用EcoR I和BamH I双酶切测序正确的pMD18-S100A1,与经同样酶切的pBV220质粒用T4 DNA连接酶连接,构建表达质粒pBV220 -S100A1,并将其转化到表达宿主E.coli DH5α中,PCR筛选阳性转化子。
4.将阳性单菌接种于含氨苄西林的LB培养基中,热激诱导表达人源S100A1蛋白。
5. SDS-PAGE和Western Blotting分析鉴定重组蛋白。
6.采用离子交换层析法纯化人源S100A1蛋白。
7.脱盐并冻干人源S100A1蛋白。
结果:
1.全基因合成了人源S100A1基因,成功构建重组表达质粒pBV220-S100A1,经酶切鉴定和DNA序列测序分析表明,该质粒含有人源S100A1基因的编码序列全长,开放阅读框架正确。
2.重组表达质粒pBV220-S100A1转化入表达宿主,诱导表达产物经纯化、SDS-PAGE和Western Blotting分析,表明表达蛋白为人源S100A1蛋白。
结论:
1.成功构建含有人源S100A1编码序列原核表达载体pBV220-S100A1。
2.经热激诱导表达、离子交换层析纯化获得纯度达95%的重组S100A1蛋白。
实验二人源S100B全基因合成和优化、表达、纯化与鉴定
方法:
1.优化人源S100B基因序列,设计合成人源S100B序列引物,在两端引物分别引入Nde I和XhoⅠ的酶切位点,分段合成法PCR合成人源S100B基因。
2.回收纯化PCR产物,TA克隆至载体pMD18-T,构建克隆载体pMD18-S100B,并将其转化到E. coli DH5α中,PCR筛选阳性转化子,小量提取质粒,经酶切鉴定后送invitrogen公司测序。
3.用Nde I和XhoⅠ双酶切测序正确的pMD18-S100B,与经同样酶切的pET32a质粒用T4 DNA连接酶连接,构建表达质粒pET32a-S100B,并将其转化到表达宿主E.coli BL21(DE3)中,PCR筛选阳性转化子。
4.将阳性单菌接种于含氨苄西林的LB培养基中,IPTG诱导表达人源S100B蛋白。
5. SDS-PAGE和Western Blotting分析鉴定重组蛋白。
6.采用离子交换层析法纯化人源S100B蛋白。
7.脱盐并冻干人源S100B蛋白。
结果:
1.全基因合成人源S100B基因,成功构建重组表达质粒pET32a-S100B,经酶切鉴定和DNA序列测序分析表明,该质粒含有人源S100B基因的编码序列全长,开放阅读框架正确。
2.重组表达质粒pET32a-S100B转化入表达宿主,诱导表达产物经纯化、SDS-PAGE和Western Blotting分析,表明表达蛋白为人源S100B蛋白。
结论:
1.成功构建含有人源S100B编码序列原核表达载体pET32a-S100B。
2.经IPTG诱导表达、离子交换层析纯化获得纯度≥95%的重组S100B蛋白。
第二部分:人源S100A1和S100B蛋白促进Hela细胞侵袭迁移研究
方法:
利用人工基底膜和基质胶,模拟在体的基质膜屏障,根据肿瘤细胞穿过基质膜的数量间接反映它的侵袭能力,而肿瘤细胞穿过不含基质胶滤膜的数量间接反映它的迁移能力。铺有基质胶的滤膜放在上下室之间,铺有基质胶面朝向上室,在下室中加趋化剂,对照组上室加入100uL重悬的Hela细胞和100uL基础培养基,实验组上室加入100uL重悬的Hela细胞和100uL重组蛋白,具有侵袭能力的细胞在趋化剂诱导下开始穿膜运动。穿过滤膜的细胞多数粘附在滤膜下表面,可用棉签将上表面的细胞拭去,然后经固定、染色,观察统计穿过基质胶的细胞数量。
结果:
1.人源S100A1蛋白显著提高Hela细胞迁移能力(与对照组相比,P<0.05),但对其侵袭能力无显著影响。
2.人源S100B蛋白可使Hela细胞的侵袭能力提高2.8倍,迁移能力提高3.5倍。
结论:
1.纯化获得的重组人源S100A1蛋白具有生物活性,可提高肿瘤细胞的迁移能力。但对其侵袭能力影响较小。
2.纯化获得的重组人源S100B蛋白具有生物活性,可提高肿瘤细胞的侵袭和迁移能力。
第三部分:抗人S100A1和S100B蛋白单克隆抗体的制备、纯化与鉴定
实验一抗人S100A1蛋白单克隆抗体的制备、纯化与鉴定
方法:
1.将纯化的重组人源S100A1蛋白和免疫佐剂共同制备成免疫原,免疫小鼠。
2.用传统的细胞融合法,将免疫成功的小鼠脾细胞与骨髓瘤细胞融合得到杂交瘤细胞。
3. ELISA检测杂交瘤细胞上清液中抗人S100A1抗体的效价,筛选阳性细胞株。
4.有限稀释法对阳性细胞株进行亚克隆,得到能够分泌目标抗体的单细胞株。
5.冻存阳性细胞株,三个月后复苏细胞,检测其抗体分泌的稳定性。
6.体内接种杂交瘤单细胞株,制备腹水型单克隆抗体,用Protein G HP层析纯化蛋白,得到人源S100A1单克隆抗体。
7. Western Blotting及ELSIA法鉴定抗体特异性。
8. ELSIA法测定抗体的亲和常数。
结果:
1.获得了4株能稳定分泌抗人S100A1单克隆抗体细胞株。
2. 4株细胞抗体亲和常数均较高,结合抗原的能力均较强,与其他抗原无交叉反应,特异性好。
结论:
1.建立了能稳定分泌抗人S100A1单克隆抗体的杂交瘤细胞株。
2.制备出抗人S100A1单克隆抗体,并进行了相关性质鉴定。
实验二抗人S100B蛋白单克隆抗体制备、纯化与鉴定
方法:
1.将纯化的重组人源S100B蛋白和免疫佐剂共同制备成免疫原,免疫小鼠。
2.用传统的细胞融合法,将免疫成功的小鼠脾细胞与骨髓瘤细胞融合得到杂交瘤细胞。
3. ELISA检测杂交瘤细胞上清液中人源S100B抗体的效价,筛选阳性细胞株。
4.有限稀释法对阳性细胞株进行亚克隆,获得能分泌目标抗体的单细胞株。
5.冻存阳性细胞株,三个月后复苏细胞,检测其抗体分泌的稳定性。
6.体内接种杂交瘤单细胞株,制备腹水型单克隆抗体,用Protein G HP纯化蛋白,获得人源S100B单克隆抗体。
7. Western Blotting及ELSIA法鉴定抗体特异性。
8. ELSIA法测定抗体的亲和常数。
结果:
1.获得了3株能稳定分泌抗人S100B单克隆抗体的细胞株。
2. 3株细胞抗体亲和常数均较高,结合抗原的能力均较强,与其它抗原无交叉反应,特异性好。
结论:
1.建立了能稳定分泌抗人S100B单克隆抗体的杂交瘤细胞株。
2.制备出抗人S100B单克隆抗体,并进行了相关特性鉴定。
第四部分:抗人S100A1和S100B单克隆抗体活性分析
方法:
1.外源性给予抗人S100A1单克隆抗体,观察其对人黑色素瘤细胞A375增殖、凋亡及P53蛋白表达的影响。
2.以商业化的抗人S100A1单克隆抗体为对照,利用制备的抗人S100A1单克隆抗体Western Blotting法检测人黑色素瘤细胞A375中S100A1蛋白的表达。
3.外源性给予抗人S100B单克隆抗体,观察其对人黑色素瘤细胞A375增殖、凋亡及P53蛋白表达的影响。
4.商业化的抗人S100B单克隆抗体为对照,利用制备的抗人S100B单克隆抗体Western Blotting法检测人黑色素瘤细胞A375中S100B蛋白的表达。
结果:
1.外源性给予抗人S100A1单克隆抗体,细胞增殖速度减慢,细胞凋亡增加,胞浆内S100A1蛋白表达降低,P53蛋白表达增多。
2. Western Blotting检测显示,S100A1蛋白在人黑色素瘤细胞A375中弱表达,且与商业化的进口单抗相比,检测效果无明显差异。
3.外源性给予抗人S100B单克隆抗体,细胞增殖速度减慢,细胞凋亡增加,胞浆内S100B蛋白表达降低,P53蛋白表达增多。
4. Western Blotting检测显示,S100B蛋白在人黑色素瘤细胞A375中有表达,但与商业化的进口单抗相比,检测效果无明显差异。
结论:
1.制备的抗人S100A1和S100B单克隆抗体既可以用于肿瘤细胞的Western Blotting检测,又可发挥抑制肿瘤细胞的增殖,起到靶向治疗作用,为相关疾病的诊断和治疗提供了新的筛查手段。
2.抗人S100A1和S100B单克隆抗体对癌细胞的靶向治疗作用,其机制可能是通过与S100A1和S100B蛋白结合,使胞浆内S100A1和S100B蛋白表达减少,增加了野生型P53蛋白表达,从而促细胞凋亡,达到抑制肿瘤细胞增殖的作用。
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 proteins
Experiment I: The whole gene synthesis and optimization, expression, purification and identification of human S100A1 protein
Methods:
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 protein
Methods:
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 stained
Results:
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 identification
Methods:
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 identification
Methods:
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.
引文
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