重组灵芝免疫调节蛋白（rLZ-8）单克隆抗体的制备及鉴定
发布时间：2018-07-31 12:30
【摘要】： LZ-8 (真菌灵芝免疫调节蛋白)是1989年Kino等人从赤灵芝子实体中分离提取出的第一种真菌免疫调节蛋白。氨基酸测序结果表明LZ-8灵芝免疫调节蛋白由110个氨基酸组成,分子量12.4KD没有组氨酸(His)、半胱氨酸(Cys)和甲硫氨酸(Met),但是富含天冬氨酸(Asp)和撷氨酸(Val)。LZ-8蛋白的二级结构中包括了7个β折叠,2个α螺旋和一个β转角,N端有大约13个氨基酸形成的α螺旋对真菌免疫调节蛋白二聚体的形成以及行使其免疫调节功能十分必要[3]。该蛋白与鼠和人的免疫球蛋白IgGA重链可变区有28%的同源性,空间结构上也有一定的相似性,IgA重链可变区富含有9个β折叠,也需要单体之间形成二聚体行使功能。 以往的研究表明,LZ-8具有促进细胞分裂和免疫调节功能,降低或完全抑制BSA(牛血清白蛋白)引起的全身免疫排斥反应,而且LZ-8对Arthus反应(一种局部过敏反应)引起的鼠足底水肿有明显的抑制作用,此外有研究人员发现LZ-8对癌细胞具有杀伤作用,认为IL-2、IL-4、TNF和IFN等几种细胞因子的表达升高以及由LC3细胞信号通路引起的自噬是抑制肿瘤细胞生长的主要原因。 本实验是根据酵母密码子偏好性将LZ-8蛋白质基因序列重新编码并命名为rLZ-8(重组灵芝免疫调节蛋白),与酵母基因组整合后诱导其表达,纯化后的蛋白质作为免疫原反复免疫小鼠。经过一定时间的培养后,取血清中能稳定表达抗LZ-8抗体的脾细胞与小鼠骨髓瘤细胞融合并通过Elisa方法筛选阳性杂交瘤细胞。选择22个最好的阳性克隆进行WB复筛,随后保留5个阳性克隆,每个克隆进行1次96孔板的亚克隆,五块版中再次挑选效价较高的亚克隆进行二次克隆,保留最好的两株杂交瘤细胞建株并进行稳定性实验、亚型鉴定、扩大培养并制备腹水、纯化抗体进行夹心抗体检测。 rLZ-8单克隆抗体的获得对今后蛋白质作用机理的研究具有深远影响,可以利用抗体和抗原分子表位(即抗原决定簇)特异性结合的原理将抗体作为探针从分子、细胞、器官等不同水平上了解蛋白质与受体相互作用、结构与功能之间关系,进而从理论上阐明作用机理,另外还可以将蛋白特异性抗体固定化便于对LZ-8的纯化。
[Abstract]:LZ-8 (fungal Ganoderma lucidum immunoregulatory protein) is the first fungal immunoregulatory protein isolated from the fruiting body of Ganoderma lucidum in 1989. The amino acid sequencing results showed that the LZ-8 Ganoderma lucidum immunoregulatory was composed of 110 amino acids, the molecular weight 12.4KD was His, cysteine (Cys) and methionine (Met), but it was rich in the amino acid (Cys) and methionine (Met). The two grade structure of aspartic acid (Asp) and ammonia (Val).LZ-8 protein consists of 7 beta folds, 2 alpha helices and a beta angle, and the N terminal has an alpha helix formed by about 13 amino acids, which is necessary for the formation of the fungal immunoregulatory protein two polymer and the exercise of its immune regulation function, [3]. and the IgGA weight of the mouse and human immunoglobulin. The variable region of the chain has 28% homology and a certain similarity in spatial structure. The variable region of IgA heavy chain is rich in 9 beta folds, and it also requires the formation of two polymers between the monomers.
Previous studies have shown that LZ-8 has the function of promoting cell division and immunoregulation, reducing or completely inhibiting the systemic immune rejection caused by BSA (bovine serum albumin), and LZ-8 has an obvious inhibitory effect on the foot edema caused by the Arthus reaction (a local anaphylaxis). In addition, researchers have found that LZ-8 has the effect of LZ-8 on the cancer cells. The increase of several cytokines, such as IL-2, IL-4, TNF and IFN, as well as autophagy caused by LC3 cell signaling pathway, is the main reason for inhibiting the growth of tumor cells.
In this experiment, the LZ-8 protein gene sequence was re coded and named as rLZ-8 (recombinant Ganoderma lucidum immunoregulatory protein) based on the preference of yeast codon, which was integrated with the yeast genome to induce its expression. The purified protein was immunized repeatedly in mice. After a certain period of culture, the anti LZ-8 resistance could be expressed stably in the serum. The spleen cells of the body were fused with the murine myeloma cells and screened the positive hybridoma cells through the Elisa method. 22 best positive clones were selected for WB rescreening, and then 5 positive clones were retained, each clone was subcloned for 1 96 orifice plates, and two clones were selected for the five best subclone in the five plate to retain the best two. The hybridoma cell lines were established and tested for stability, subtype identification, enlarged culture and preparation of ascites, purified antibody for sandwich antibody detection.
The acquisition of rLZ-8 monoclonal antibodies has a profound influence on the mechanism of protein action in the future. It is possible to use the principle of antibody and antigen molecular epitope specific binding to understand the interaction between protein-protein and receptor, structure and function as a probe at different levels of molecules, cells and organs. Furthermore, the mechanism of action can be elucidated theoretically, and the specific antibody of protein can be immobilized to facilitate the purification of LZ-8.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2155590
[Abstract]:LZ-8 (fungal Ganoderma lucidum immunoregulatory protein) is the first fungal immunoregulatory protein isolated from the fruiting body of Ganoderma lucidum in 1989. The amino acid sequencing results showed that the LZ-8 Ganoderma lucidum immunoregulatory was composed of 110 amino acids, the molecular weight 12.4KD was His, cysteine (Cys) and methionine (Met), but it was rich in the amino acid (Cys) and methionine (Met). The two grade structure of aspartic acid (Asp) and ammonia (Val).LZ-8 protein consists of 7 beta folds, 2 alpha helices and a beta angle, and the N terminal has an alpha helix formed by about 13 amino acids, which is necessary for the formation of the fungal immunoregulatory protein two polymer and the exercise of its immune regulation function, [3]. and the IgGA weight of the mouse and human immunoglobulin. The variable region of the chain has 28% homology and a certain similarity in spatial structure. The variable region of IgA heavy chain is rich in 9 beta folds, and it also requires the formation of two polymers between the monomers.
Previous studies have shown that LZ-8 has the function of promoting cell division and immunoregulation, reducing or completely inhibiting the systemic immune rejection caused by BSA (bovine serum albumin), and LZ-8 has an obvious inhibitory effect on the foot edema caused by the Arthus reaction (a local anaphylaxis). In addition, researchers have found that LZ-8 has the effect of LZ-8 on the cancer cells. The increase of several cytokines, such as IL-2, IL-4, TNF and IFN, as well as autophagy caused by LC3 cell signaling pathway, is the main reason for inhibiting the growth of tumor cells.
In this experiment, the LZ-8 protein gene sequence was re coded and named as rLZ-8 (recombinant Ganoderma lucidum immunoregulatory protein) based on the preference of yeast codon, which was integrated with the yeast genome to induce its expression. The purified protein was immunized repeatedly in mice. After a certain period of culture, the anti LZ-8 resistance could be expressed stably in the serum. The spleen cells of the body were fused with the murine myeloma cells and screened the positive hybridoma cells through the Elisa method. 22 best positive clones were selected for WB rescreening, and then 5 positive clones were retained, each clone was subcloned for 1 96 orifice plates, and two clones were selected for the five best subclone in the five plate to retain the best two. The hybridoma cell lines were established and tested for stability, subtype identification, enlarged culture and preparation of ascites, purified antibody for sandwich antibody detection.
The acquisition of rLZ-8 monoclonal antibodies has a profound influence on the mechanism of protein action in the future. It is possible to use the principle of antibody and antigen molecular epitope specific binding to understand the interaction between protein-protein and receptor, structure and function as a probe at different levels of molecules, cells and organs. Furthermore, the mechanism of action can be elucidated theoretically, and the specific antibody of protein can be immobilized to facilitate the purification of LZ-8.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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