镁离子是一种广泛使用的海洋软体动物肌肉松弛剂，获取方便、性质稳定、毒副作用小。在高剂量镁离子作用下，牡蛎发生闭壳肌松弛并丧失对外界刺激的反应，这一过程至少涉及平滑肌和横纹肌两种类型的肌肉松弛，与脊椎动物中高剂量镁离子仅能使平滑肌松弛的表现十分不同。该差异体现了有代表性的、值得关注的神经肌肉作用方式，因此深入讨论其背后的分子机制十分必要，目前有关该机制的研究仍属空白。本文以长牡蛎（Crassostrea gigas）为研究对象，围绕镁离子使长牡蛎闭壳肌发生松弛这一生理现象背后的机制分别在个体水平和分子水平展开初步探究，主要研究内容与结果如下：

1）对镁离子使长牡蛎闭壳肌发生松弛的生理现象进行验证，对比分析部分其他一价、二价、三价金属离子是否会对长牡蛎闭壳肌产生相似的松弛作用。实验结果表明，与其他金属离子相比，镁离子使长牡蛎闭壳肌松弛的作用十分有效，30 g/L是较优的镁离子处理浓度，浸泡处理45 min即可使80%的长牡蛎发生闭壳肌松弛；降低镁离子浓度至15 g/L或7.5 g/L时，处理时间足够长的前提下也可使全部长牡蛎发生闭壳肌松弛。本研究实验中采用的Mg²⁺浓度均未对长牡蛎的生存造成不利影响。在其他金属离子，如一价离子Li⁺、Na⁺、K⁺，二价离子Co²⁺、Mn²⁺、Sr²⁺，三价离子Fe³⁺中，只有MnCl₂和SrCl₂体系对长牡蛎表现出微弱的闭壳肌松弛作用。

2）探究了镁离子造成长牡蛎闭壳肌松弛过程中钙离子的拮抗作用。设计不同浓度镁离子/钙离子共存的溶液体系，以及镁离子/钙离子/EGTA溶液体系，观察并统计长牡蛎的生理状态变化。结果显示，随着镁钙共存体系中钙离子浓度的升高，发生闭壳肌松弛的长牡蛎个体随之减少，当镁钙摩尔比为2:5时，90%的长牡蛎样本无法产生闭壳肌松弛，并且MgCl₂溶液处理后发生闭壳肌松弛的长牡蛎可在该体系中恢复至正常生理状态，用EGTA螯合全部钙离子后，长牡蛎再次发生闭壳肌松弛。以上数据说明镁离子对长牡蛎的闭壳肌松弛作用会受到高剂量钙离子的抑制。

3）钙离子通道在肌肉运动过程中发挥了重要作用。依据前述生理实验结果，并结合脊椎动物中镁离子可在电压门控钙离子通道（VGCC）上拮抗钙离子的相关研究，本论文在分子水平的实验主要定位在长牡蛎闭壳肌VGCC复合体上。基于β亚基与α1亚基之间较强的亲和力，将原核表达纯化的重组β亚基用于VGCC复合体的pull down纯化。完成了长牡蛎闭壳肌中VGCC β亚基的克隆，构建了GST-β、β-His、His-β等多种不同表达载体，对β亚基进行原核表达及纯化条件优化，结果表明GST-β亚基的纯度和产量更高；将纯化的GST-β亚基用于VGCC复合体的pull down纯化，未获得足量的高纯度VGCC复合体。同时，经过序列分析，发现β亚基具有多组组氨酸残基聚集的区域，制备并纯化了无标签的野生型β亚基重组蛋白。相比于GST-β亚基，借助无标签亲和层析纯化得到的野生型β亚基重组蛋白为单体且纯度更高。无标签亲和层析纯化的方法可用于长牡蛎VGCC复合体的提取纯化及各步骤的监测。

4）通过突变实验，探究了无标签亲和层析纯化方法的序列基础。发现重组蛋白对Ni-NTA的结合能力与剩余较连续组氨酸残基的数量呈正相关。免疫印迹对组氨酸残基的数量及连续性的要求更高，(HG)₇序列是组氨酸标签特异性抗体识别的关键序列。

本论文主要关注镁离子使长牡蛎闭壳肌发生松弛这一生理现象，提出了其背后机制是镁离子在VGCC上对钙离子的拮抗，并开展了VGCC关键亚基的表达纯化和VGCC复合体的初步提取研究，研究结果为后续长牡蛎VGCC复合体的结构解析提供了必不可少的研究基础，为阐释以长牡蛎为代表的软体动物的神经肌肉活动调控机制提供了参考。同时，无标签亲和层析纯化方法为具有相似特点的蛋白纯化提供了借鉴，也为解决缺乏特异性抗体的蛋白复合体的分离纯化监测难题贡献了新思路。

Magnesium ion is a widely used muscle relaxant for marine mollusks, which is easy to obtain, stable and little side effect. The adductor muscle of oysters relaxes and loses its response to external stimuli with high-dose Mg²⁺. This process involves at least two types of muscle relaxation, namely smooth muscle and striated muscle, which is quite different from the performance of high-dose Mg²⁺ in vertebrates, which can only make smooth muscle relax. The difference reflects a representative and noteworthy mode of neuromuscular action, so it is necessary to discuss the molecular mechanism in depth. Currently, research on this mechanism is still blank. Taking Pacific oyster (Crassostrea gigas) as the research object, this paper carried out a preliminary study at the individual and molecular levels on the mechanism behind the physiological phenomenon that magnesium ions relax the adductor muscle of oysters. The main research contents and results are as follows:

1) Verify the physiological phenomenon that magnesium ions relax the adductor muscle of oyster, compare and analyze whether some other monovalent, divalent and trivalent metal ions will have a similar relaxation effect on oyster adductor muscle. The experimental results showed that, compared with other metal ions, magnesium ions were very effective in relaxing the adductor muscle of oysters. 80% of oyster adductor muscle relaxation occurred after soaking in the optimal treatment concentration of 30 g/L magnesium ions for 45 min. When the concentration of magnesium ion was reduced to 15 g/L or 7.5 g/L, the adductor muscle relaxation could also occur in all oysters under the premise that the treatment time was long enough. None of the experimental Mg²⁺ concentration used in this study had adverse effects on the survival of oysters. Among other metal ions such as monovalent ions Li⁺, Na⁺, K⁺, divalent ions Co²⁺, Mn²⁺, Sr²⁺, trivalent ions Fe³⁺, only MnCl₂ and SrCl₂ system showed weak adductor muscle relaxation effect on oysters.

2) The antagonistic effect of calcium ions on the relaxation of adductor muscle induced by magnesium was investigated. The changes in the physiological state of oysters were observed and recorded, when treated with different concentrations of magnesium ion/calcium ion coexistence system or magnesium ion/calcium ion/EGTA solution system. The results showed that with the increase of calcium ion concentration in the magnesium-calcium coexistence system, the number of oyster individuals with adductor muscle relaxation decreased. When the magnesium-calcium molar ratio was 2:5, adductor muscle relaxation in 90% of the oyster samples could not occur. And the oysters whose adductor muscle relaxed after treatment with MgCl₂ solution could recover to normal physiological state in this system. After chelating all calcium ions with EGTA, the adductor muscle relaxation occurred again. The above data indicated that the relaxation effect of magnesium ions on oyster adductor muscle was inhibited by high doses of calcium ions.

3) Calcium channels play an important role in muscle movement. Based on the above physiological experimental results, and combined with the related research in vertebrates that magnesium ions can antagonize calcium ions on the voltage-gated calcium channel (VGCC), the molecular-level experiments in this paper are mainly focused on the VGCC complex in adductor muscle of Crassostrea gigas. Based on the strong affinity between the β subunit and the α1 subunit, the recombinant β subunit with prokaryotic expression and purification was used for the pull down purification of the VGCC complex. The VGCC β subunit was cloned from adductor muscle of Crassostrea gigas and a variety of different expression vectors such as GST-β, β-His and His-β were constructed. The prokaryotic expression and purification conditions of β subunit were optimized. The results showed that the purity and yield of the GST-β subunit were better. The purified GST-β subunit was used for pull down purification of VGCC complex, but a sufficient amount of high-purity VGCC complex was not obtained. At the same time, after sequence analysis, it was found that the β subunit had regions where multiple histidine residues were aggregated, and the wild-type β subunit recombinant protein without affinity tag was prepared and purified. Compared with GST-β subunit, the wild-type β subunit recombinant protein with homogeneity and higher purity was obtained through tag-free affinity chromatography. The tag-free affinity chromatography purification method can be used for the extraction and purification monitoring of the VGCC complex in Crassostrea gigas.

4) Through mutation experiments, the sequence basis of the tag-free affinity chromatography purification method was explored. It was found that the binding ability of recombinant protein to Ni-NTA was positively correlated with the number of continuous histidine residues. Western blot had higher requirements on the number and continuity of histidine residues, and the (HG)₇ sequence was key to the histidine tag-specific antibody recognition.

In this study, we focused on the physiological phenomenon that magnesium ions relax oyster adductor muscle, and proposed that the mechanism is the antagonism of magnesium ion to calcium ion on VGCC, and carried out the expression and purification of key subunits of VGCC and the preliminary extraction of VGCC complex. The results of this paper provide an essential research basis for the subsequent structural analysis of oyster VGCC complex, and provide a reference for the interpretation of the neuromuscular activity regulation mechanism in mollusks represented by oyster. In the meantime, the tag-free affinity chromatography purification method provides a reference for the purification of proteins with similar characteristics, and also contributes new ideas to solve the problem of monitoring the separation and purification of protein complex lacking specific antibodies.

第一章  绪论.... 1

1.1  以牡蛎为代表的软体动物肌肉松弛的研究背景... 1

1.2  镁离子在神经肌肉活动中的生理作用... 3

1.2.1  镁离子在心血管系统中的作用... 4

1.2.2  镁离子在神经系统中的作用... 4

1.2.3  镁离子在肌肉中的作用... 5

1.3  离子通道概述... 6

1.4  蛋白质提取纯化方法概述... 11

1.5  课题研究意义及内容... 13

1.5.1  研究意义... 13

1.5.2  研究内容与技术路线... 14

第二章  长牡蛎闭壳肌松弛作用的个体水平生理研究.... 17

2.1  前言... 17

2.2  材料与方法... 17

2.2.1  实验所用试剂... 17

2.2.2  不同溶液体系的配制... 17

2.2.3  长牡蛎闭壳肌松弛的观察与统计... 18

2.3  结果... 19

2.3.1  镁离子在长牡蛎闭壳肌松弛过程中的作用... 19

2.3.2  其他金属离子在长牡蛎闭壳肌松弛过程中的作用... 20

2.3.3  镁钙共存体系中长牡蛎的闭壳肌松弛... 22

2.3.4  Mg-Ca-EGTA体系中长牡蛎的闭壳肌松弛... 24

2.4  讨论... 25

第三章  长牡蛎闭壳肌VGCC β亚基的克隆、表达与纯化.... 27

3.1  前言... 27

3.2  材料与方法... 28

3.2.1  主要仪器设备... 28

3.2.2  长牡蛎闭壳肌总RNA的提取... 28

3.2.3  VGCC β亚基的克隆... 29

3.2.4  长牡蛎β亚基表达载体的构建... 29

3.2.5  长牡蛎β亚基的诱导表达... 31

3.2.6  优化外源GST标签融合β亚基的纯化条件... 32

3.2.7  优化外源His标签融合β亚基的纯化条件... 33

3.2.8  长牡蛎野生型β亚基的无标签亲和层析纯化... 33

3.2.9  基于SEC-MALS技术的野生型β亚基聚集状态分析... 33

3.2.10  长牡蛎β亚基的聚丙烯酰胺凝胶电泳和免疫印迹分析... 34

3.2.11  长牡蛎VGCC复合体的原位粗提取... 35

3.2.12  基于GST-β亚基的VGCC复合体pull down纯化... 35

3.2.13  长牡蛎VGCC复合体的无标签亲和层析纯化... 36

3.3  结果... 36

3.3.1  外源GST标签融合β亚基的纯化条件优化... 36

3.3.2  外源His标签融合β亚基的纯化条件优化... 43

3.3.3  无标签野生型β亚基纯化... 44

3.3.4  GST-β亚基对长牡蛎VGCC复合体的pull down纯化... 46

3.3.5  长牡蛎VGCC复合体原位粗提取及监测... 47

3.3.6  无标签亲和层析纯化长牡蛎VGCC复合体... 49

3.4  讨论... 50

第四章  长牡蛎β亚基无标签亲和层析纯化方法的序列基础探究.... 53

4.1  前言... 53

4.2  材料与方法... 53

4.2.1  主要仪器设备... 53

4.2.2  长牡蛎VGCC β亚基序列分析... 54

4.2.3  无亲和标签的野生型β亚基表达载体构建... 54

4.2.4  突变型β亚基表达载体的构建... 54

4.2.5  β亚基的诱导表达... 55

4.2.6  野生型及突变型β亚基的纯化... 56

4.2.7  聚丙烯酰胺凝胶电泳分析及免疫印迹鉴定... 57

4.3  结果... 57

4.3.1  长牡蛎VGCC β亚基序列分析... 57

4.3.2  不同亲和柱的选择与分析... 60

4.3.3  野生型及突变型β亚基的纯化... 61

4.4  讨论... 64

第五章  结论与展望.... 65

5.1  结论... 65

5.2  展望... 66

参考文献.... 69

致  谢.... 79

作者简历及攻读学位期间发表的学术论文与研究成果    81