摘要
南瓜胰蛋白酶抑制剂I(Cucurbita maxima trypsin inhibitor I,CMTI-I)是一种来源于南瓜籽的经典类丝氨酸蛋白酶抑制剂,该家族由20多种成员组成。CMTI的成员分子量小、结构简单、作用机制清楚,是研究活性蛋白质分子结构与功能关系及作用机制的良好工具。由于CMTI家族各成员在分子量和结构方面相近,目前很难从南瓜籽中分离纯化出大量单一CMTI,从而限制了其药用开发研究。为此,我室构建了表达重组CMTI-I(rCMTI-I)的GSll5毕赤酵母工程菌。为了对rCMTI-I进行深入的研究与开发,本论文研究制备了以微晶纤维素为固定化介质的rCMTI-I亲和纯化介质,建立了rCMTI-I的分离纯化工艺,研究了rCMTI-I对肿瘤细胞增殖的影响,并研究了rCMTI-I对明胶酶的作用。研究内容与结果主要有以下几个方面。
1.rCMTI-I亲和层析介质的制备
首先用环氧氯丙烷活化柱层析用微晶纤维素,并以胰蛋白酶为配基交联于其上获得了亲和层析介质,同时考察了微晶纤维素的最佳活化条件以及制备微晶纤维素亲和层析介质最佳胰蛋白酶用量。最佳活化条件为1:1的2.4mol/L NaOH溶液与DMSO(体积比)作为混合溶剂,活化温度为30℃,活化时间3h环氧氯丙烷的用量为20ml/g时环氧基密度最大。在最佳活化条件下,每克微晶纤维素的环氧基含量为(302.4908±10)μmol/g。
2.rCMTI-1分离纯化工艺的研究
用所制备的亲和层析介质从含有rCMTI-I的培养基上清中直接分离纯化rCMTI-I。通过Sephadex G-10层析对获得的亲和层析洗脱液冷冻干燥品进行脱盐,再用Sephadex G-25层析进一步纯化,采用Tricine-SDS PAGE以及抑制活力测定对活性蛋白峰进行追踪,获得了基本纯化的rCMTI-I。与用阳离子交换剂和阴离子交换剂作为纯化介质相比,亲和层析介质纯化效果较好。
3.rCMTI-I对肿瘤细胞增殖的影响
研究了rCMTI-I对肿瘤细胞增殖的影响,发现rCMTI-I对卵巢癌SKOV3细胞和SE-2肿瘤细胞的增殖有非常明显的抑制作用,并且这种抑制呈明显的剂量—效应关系,在浓度为3.82×10~2μmol/ml时rCMTI-I对两种肿瘤细胞的增殖抑制率分别为69.3%和86.5%。本结果预示着rCMTI-I有抗肿瘤作用。
4.rCMTI-I对肿瘤细胞增殖的影响
采用琥珀酰明胶法测定rCMTI-I对明胶酶的作用,显示rCMTI-I具有抑制明胶酶的活性,揭示rCMTI-I可能具有抑制明胶酶降解基底膜抑制肿瘤转移的作用。
本研究取得的主要研究成果有:
(1)研究制备了偶联有胰蛋白酶的柱层析用微晶纤维素亲和层析介质,并在此基础上研究建立了从工程菌发酵表达的上清液中分离纯化rCMTI-I的工艺,该工艺适合规模化生产。
(2)研究发现rCMTI-I具有抑制肿瘤细胞增殖的活性,揭示rCMTI-I可能具有抗肿瘤作用。
(3)采用琥珀酰明胶法测定rCMTI-I对明胶酶的作用,显示rCMTI-I具有抑制明胶酶的活性,揭示rCMTI-I可能具有抑制明胶酶降解基底膜抑制肿瘤转移的作用。
本研究对小分子活性肽的表达、分离纯化具有指导意义,为rCMTI-I的药用开发奠定了基础。
Cucurbita maxima trypsin inhibitor I (CMTI-I) is one kind of the serine protease inhibitors from Cucurbita family which consists of more than 20 members. Because of their low molecular weights, simple structures and clear mechanism, CMTIs are used as tools for the study of the structure-function relationships of active proteins and their mechanism. Though natural CMTIs have clear activities, it is difficult to develope them into drugs because of their similarities in molecular weights and structures of different members of the family, which results in the difficulty to get large amount of CMTI-I by isolation from Cucurbita maxima seeds. For this reason, the genetic engineering strains of Pichia pastoris GS115 producing recombinant CMTI-I (rCMTI-I) was constructed in our lab. In order to carry out deep research and development for rCMTI-I, the isolation and purification procedure was established and the tumor cell multiplication inhibition activity was studied in this paper. The main research contents and results are as follows.
1 Preparation of rCMTI-I affinity chromatography medium
In order to establish a cheap isolation and purification procedure for rCMTI-I by affinity chromatography, the preparation of the affinity medium was studied. First, the affinity medium was prepared by activating microcry stalline cellulose for column chromatography use with epichlorohydrin and coupling trypsin as the lingand to the matrix. Meanwhile activating conditions and the use of trypsin for the affinity medium were studied. The best condition for activation is using a mixture solvent of 2.4mol/L NaOH solution and DMSO(volume rate is 1: 1), at 30℃, activating for 3 hours, using 20ml of epichlorohydrin for microcry stalline cellulose of 1g. The epoxy density of activated microcry stalline cellulose under the best condition was (302.4908±10)μmol/g.
2 Establishment of rCMTI-I purification procedure
Rcombiment CMTI-I was isolated directly from the culture medium by using the prepared affinity medium. The resulting rCMTI-I sample was desalted by Sephadex G-10 which could remove the low molecular weight materials of rCMTI-I and salt at the same time and further purified by Sephadex G-25 chromatography. Pure rCMTI-I was obtained. Affinity chromatography was more effective compared with ion exchange chromatography.
3 Study on the tumor cell multiplication inhibition activity of rCMTI-I
The in vitro study of tumor cell multiplication inhibition activity of rCMTI-I showed that rCMTI-I could significantly inhibit SKOV3 cell line and SE-2 cell line, and the action had a dose-dependent effect with inhibition rates of 69.3% and 86.5% for the two cell lines respectively at the concentration of 3.82×10~(-2)μmol/ml. The results suggest that rCMTI-I may have anti-tumor activity.
4 Study on the MMPs inhibition activity of rCMTI-I
The inhibition activity of rCMTI-I on MMPs was tested with TNBSA assay, which showed a dose-dependent effect. The results suggest that rCMTI-I may have anti-tumor metastasis effect.
The main achievements obtained in the study are as follows:
a. The isolation and purification procedure of rCMTI-I from the supernatant of cultured medium was established on the basis of affinity medium preparation and investigation. The procedure is suitable for large scale production.
b. The tumor cell multiplication inhibition activity on tumor cells of rCMTI-I was found, which gave us a new thought to study the anti-tumor activity of rCMTI-I.
c. The activity of rCMTI-I on MMPs was tested with TNBSA assay, which showed an inhibition activity.
The study has laid the foundation for the development of rCMTI-I as a new drug.
引文
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