大鼠粘蛋白rMuc3酶切保守基序对其蛋白酶切的影响的研究

发布时间：2019-02-19 09:21

【摘要】： 目的:通过定点突变技术得到保守基序的各种突变体(p201/a、p20s_1/a、p20k/a、p20i/a、p20v_1/a、p20v_2/a),并通过Western blotting探讨保守基序中各氨基酸对大鼠粘蛋白rMuc3蛋白酶切的影响。方法: 1、采用定点突变技术,设计相应突变引物,以p20为模板,基于PCR扩增得到突变体,并经测序验证。 2、用质粒中量提取试剂盒得到足量的质粒,然后利用阳离子脂质体LipofectAMINE将各突变体及p20转染入COS-7细胞中。 3、通过Western blotting检测各突变体的表达,采用Quantity one分析各种突变体未酶切和酶切部分的表达强度,并分析未酶切部分所占比例。结果: 1、使用PCR定点突变的方法,获得突变体,经序列测定后利用clustalx1.83软件与模板p20比对,证实突变完全成功。并通过质粒中量提取获得了足量的质粒用于转染。 2、裂解细胞后将细胞裂解物进行Western blotting免疫印迹实验,证实在55kDa处存在未酶切的部分,在30kDa处存在可被抗V5抗体检测的N端部分,经过Quantityone分析后发现,S_2/A完全抑制了酶切的发生,G/A抑制了绝大部分的酶切(未酶切部分占79%),L/A、I/A、V_1/A、V_2/A均对酶切产生了不同程度的抑制,未酶切部分分别为22%、39%、14%和17%,而K/A和S_1/A几乎对酶切没有影响,其未酶切部分分别为6%和3%,酶切效率与p20(未酶切部分占4%)几乎一样。结论:以p20为模板的各种突变体突变成功,以脂质体转染法将所有突变体导入COS-7细胞并获得了相应突变体的高效表达。通过Western blotting免疫印迹实验结果揭示了酶切保守基序LS_1KGS_2IV_1V_2中各氨基酸对其蛋白酶切的发生是很重要的,其机理可能是因为此保守基序位于β2和β3片层之间的环状区,其上的氨基酸对于维持粘蛋白的构象是必须的。在S_1上可能有O型糖链的存在,并且去除此O型糖链不影响酶切保守基序LS_1KGS_2IV_1V_2的酶切。
[Abstract]:Objective: to obtain various mutants with conserved motifs by site-directed mutation technique. The effects of amino acids in conserved motifs on the digestion of rat mucin rMuc3 protease were studied by Western blotting. Methods: 1. Using site-directed mutagenesis technique, the corresponding mutated primers were designed, using p20 as template, the mutants were amplified based on PCR and confirmed by sequencing. 2. A large number of plasmids were obtained by plasmid extraction kit, and then the mutants and p20 were transfected into COS-7 cells by cationic liposome LipofectAMINE. (3) the expression of the mutants was detected by Western blotting, and the expression intensity of the undigested and digested parts of the mutants was analyzed by Quantity one, and the proportion of the undigested parts was analyzed. Results: 1. By using the method of PCR site-directed mutation, the mutants were obtained. After sequencing, clustalx1.83 software and template p20 were used to confirm that the mutation was completely successful. A sufficient number of plasmids were obtained by extraction of plasmids for transfection. (2) the cell lysate was detected by Western blotting assay. The results showed that there were no enzyme digested parts in 55kDa and N-terminal parts in 30kDa which could be detected by anti-V5 antibody. After Quantityone analysis, it was found that, S2 / A completely inhibited the occurrence of enzyme digestion. G / A inhibited most of the enzyme digestion (79% of the undigested part). The undigested part was 2240% and 17%, while K / A and S / A had almost no effect on the enzyme digestion. The undigested part was 6% and 3%, respectively, and the digesting efficiency was almost the same as that of p20 (4% of the undigested part). Conclusion: all the mutants using p20 as template were successfully transformed into COS-7 cells by liposome transfection and the corresponding mutants were highly expressed. The results of Western blotting showed that the amino acids in the conserved motif LS_1KGS_2IV_1V_2 were important for the proteolysis, and the mechanism might be that the conserved motifs were located in the annular region between 尾 _ 2 and 尾 _ 3 lamellae. The amino acids on it are necessary to maintain the conformation of mucin. There may be O-type sugar chain in S-1, and the removal of O-type sugar chain does not affect the enzyme digestion of conserved motif LS_1KGS_2IV_1V_2.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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