【摘要】： LZ-8 (真菌靈芝免疫調(diào)節(jié)蛋白)是1989年Kino等人從赤靈芝子實(shí)體中分離提取出的第一種真菌免疫調(diào)節(jié)蛋白。氨基酸測(cè)序結(jié)果表明LZ-8靈芝免疫調(diào)節(jié)蛋白由110個(gè)氨基酸組成,分子量12.4KD沒(méi)有組氨酸(His)、半胱氨酸(Cys)和甲硫氨酸(Met),但是富含天冬氨酸(Asp)和擷氨酸(Val)。LZ-8蛋白的二級(jí)結(jié)構(gòu)中包括了7個(gè)β折疊,2個(gè)α螺旋和一個(gè)β轉(zhuǎn)角,N端有大約13個(gè)氨基酸形成的α螺旋對(duì)真菌免疫調(diào)節(jié)蛋白二聚體的形成以及行使其免疫調(diào)節(jié)功能十分必要[3]。該蛋白與鼠和人的免疫球蛋白IgGA重鏈可變區(qū)有28%的同源性,空間結(jié)構(gòu)上也有一定的相似性,IgA重鏈可變區(qū)富含有9個(gè)β折疊,也需要單體之間形成二聚體行使功能。 以往的研究表明,LZ-8具有促進(jìn)細(xì)胞分裂和免疫調(diào)節(jié)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應(yīng),而且LZ-8對(duì)Arthus反應(yīng)(一種局部過(guò)敏反應(yīng))引起的鼠足底水腫有明顯的抑制作用,此外有研究人員發(fā)現(xiàn)LZ-8對(duì)癌細(xì)胞具有殺傷作用,認(rèn)為IL-2、IL-4、TNF和IFN等幾種細(xì)胞因子的表達(dá)升高以及由LC3細(xì)胞信號(hào)通路引起的自噬是抑制腫瘤細(xì)胞生長(zhǎng)的主要原因。 本實(shí)驗(yàn)是根據(jù)酵母密碼子偏好性將LZ-8蛋白質(zhì)基因序列重新編碼并命名為rLZ-8(重組靈芝免疫調(diào)節(jié)蛋白),與酵母基因組整合后誘導(dǎo)其表達(dá),純化后的蛋白質(zhì)作為免疫原反復(fù)免疫小鼠。經(jīng)過(guò)一定時(shí)間的培養(yǎng)后,取血清中能穩(wěn)定表達(dá)抗LZ-8抗體的脾細(xì)胞與小鼠骨髓瘤細(xì)胞融合并通過(guò)Elisa方法篩選陽(yáng)性雜交瘤細(xì)胞。選擇22個(gè)最好的陽(yáng)性克隆進(jìn)行WB復(fù)篩,隨后保留5個(gè)陽(yáng)性克隆,每個(gè)克隆進(jìn)行1次96孔板的亞克隆,五塊版中再次挑選效價(jià)較高的亞克隆進(jìn)行二次克隆,保留最好的兩株雜交瘤細(xì)胞建株并進(jìn)行穩(wěn)定性實(shí)驗(yàn)、亞型鑒定、擴(kuò)大培養(yǎng)并制備腹水、純化抗體進(jìn)行夾心抗體檢測(cè)。 rLZ-8單克隆抗體的獲得對(duì)今后蛋白質(zhì)作用機(jī)理的研究具有深遠(yuǎn)影響,可以利用抗體和抗原分子表位(即抗原決定簇)特異性結(jié)合的原理將抗體作為探針從分子、細(xì)胞、器官等不同水平上了解蛋白質(zhì)與受體相互作用、結(jié)構(gòu)與功能之間關(guān)系,進(jìn)而從理論上闡明作用機(jī)理,另外還可以將蛋白特異性抗體固定化便于對(duì)LZ-8的純化。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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