鱼精蛋白(Protamine)抗菌特性及其编码基因的克隆与表达研究
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摘要
鱼精蛋白是一种存在于各类动物精巢核组织中的多聚阳离子肽，在精子的成熟期间逐步表达并代替组蛋白而形成的。国内外研究表明，作为一种新型的食品防腐剂，鱼精蛋白在应用上具有高效、广谱、安全等优点，是一种值得开发的天然食品防腐剂。而且，鱼精蛋白具有营养性和功能性，对人体有许多种保健作用，在医疗中也得到了广泛的应用。
本实验初步研究了一些可能的理化因素对鱼精蛋白抗菌效果的影响，为其在实际应用中提供一定的参考。另外，以微生物为宿主重组表达生物活性蛋白或多肽一直是生物技术研究的热点，本实验还通过PCR技术得到鱼精蛋白的编码基因，以大肠肝菌(E．coli)为宿主微生物重组表达鱼精蛋白。主要结果如下：
(1)食品中的Ca~(2+)、Mg~(2+)、K~+、Na~+等金属离子对鱼精蛋白抗菌活性影响较大，且呈负面影响。Ca~(2+)、Mg~(2+)、K~+、Na~+等电荷数和浓度越高，影响也越大，影响大小依次为：Ca~(2+)＞Mg~(2+)＞K~+＞Na~+。
(2)鱼精蛋白在中性和碱性条件下抗菌活性明显，而在酸性条件下效果较差。
(3)鱼精蛋白的热稳定性好，其抗菌效果受温度的影响不大，这使它可以与食品热处理并用来提高食品的保存性。
(4)鱼精蛋白的抗菌效果迅速，是一种即时效应，可以在短时间内抑制大部分微生物。
(5)食品中的有机成分如蛋白质、脂肪、糖类对鱼精蛋白抗菌活性影响较小。在3种有机质中，脂肪的影响最大。
(6)鱼精蛋白是小分子肽，有些蛋白酶如胰凝乳蛋白酶、胰蛋白酶、木瓜蛋白酶、胃蛋白酶可分解它，使其一级结构受到破坏，从而影响它的抗菌活性。
(7)通过PCR技术扩增鱼精蛋白的DNA片段，以pUCT为克隆载体克隆到了鱼精蛋白编码基因。与已知序列相比较，只存在3处变异，同源性高达97.1％。
(8)以E．coli为重组表达宿主，pET30a为表达载体，构建了鱼精蛋白的融合蛋白表达系统。IPTG诱导表达后，SDS-PAGE电泳显示实验组比对照组增加了一条具有预期分子量的亮带，初步证实了融合蛋白表达系统的成功构建。
Protamine is a kind of calionic antimicrobial pcptidc(CAP) found in the nuclei of sperm of different animals species, where it replaces histone-protamine during maturation of the spermatid. Studies indicate that, being a novel natural food preservative, protamine is effective, safe, with broad anti-microbial spectrum and worthy of further exploitation. Furthermore, protamine is beneficial to health because of its nutrition and functionality, it also has been applied broadly in medical treatment.
To provide some reference to the application of protamine, the experiment studied some physical and chemical factors which affect protamine's antimicrobial efficacy, in addition, we have cloned coding sequence of protamine by PCR, and expressed the fusion protein of protamine in E.coli. The main results were as following:
(1) Metal ions like Ca2+ Mg2+, K+% Na+ have negative effects on antimicrobial efficacy of protamine. The higher charge and concentration of metal ions, the bigger their effects on antimicrobial efficacy of protamine, and the degree hirarchy of effect was: Ca2+>Mg2+>K+>Na+.
(2) Antimicrobial efficacy of protamine is evidence under the condition of neutral and alkalescence, but it is low in the acidic condition.
(3) The temperature treatment show little effect on antimicrobial efficacy of protamine, which makes protamine ideal for application in food together with heat treatment to prolong shelf life of food.
(4) antimicrobial action of protamine is rapid and immediate effect, it can inhibit or kill majority of microorganism during a short time.
(!>) Organic substances such as protein, fat and sugar in food have a small effect on antimicrobial efficacy of protamine. among three organic substances, the effect of fat is more significant than other.
(6) Being a small peptide molecule, protamine's basic structure can be destroyed and it's antimicrobial efficacy largely affected by some protein enzymes like chymotrypsin, trypsin, papain and pepsin.
(7) A fragment of protamine DNA was cloned by PCR, and its sequence determined after cloning into a pUCT vector . Compared with known protamine gene sequence, there are three single nucleic acid variations in our senquence but their homology is up to 91.1%.
(8) we have also constructed the fusion expression system for prolamine using pET30a as expression vector and E.coli as expression strain. When induced by IPTG, the system expressed a new approximate 25 KD protain as checked by SDS-PAGE electrophoresis. we tentatively think that it is the target fusion protein of protamine combined with E.coli protein .
本实验初步研究了一些可能的理化因素对鱼精蛋白抗菌效果的影响，为其在实际应用中提供一定的参考。另外，以微生物为宿主重组表达生物活性蛋白或多肽一直是生物技术研究的热点，本实验还通过PCR技术得到鱼精蛋白的编码基因，以大肠肝菌(E．coli)为宿主微生物重组表达鱼精蛋白。主要结果如下：
(1)食品中的Ca~(2+)、Mg~(2+)、K~+、Na~+等金属离子对鱼精蛋白抗菌活性影响较大，且呈负面影响。Ca~(2+)、Mg~(2+)、K~+、Na~+等电荷数和浓度越高，影响也越大，影响大小依次为：Ca~(2+)＞Mg~(2+)＞K~+＞Na~+。
(2)鱼精蛋白在中性和碱性条件下抗菌活性明显，而在酸性条件下效果较差。
(3)鱼精蛋白的热稳定性好，其抗菌效果受温度的影响不大，这使它可以与食品热处理并用来提高食品的保存性。
(4)鱼精蛋白的抗菌效果迅速，是一种即时效应，可以在短时间内抑制大部分微生物。
(5)食品中的有机成分如蛋白质、脂肪、糖类对鱼精蛋白抗菌活性影响较小。在3种有机质中，脂肪的影响最大。
(6)鱼精蛋白是小分子肽，有些蛋白酶如胰凝乳蛋白酶、胰蛋白酶、木瓜蛋白酶、胃蛋白酶可分解它，使其一级结构受到破坏，从而影响它的抗菌活性。
(7)通过PCR技术扩增鱼精蛋白的DNA片段，以pUCT为克隆载体克隆到了鱼精蛋白编码基因。与已知序列相比较，只存在3处变异，同源性高达97.1％。
(8)以E．coli为重组表达宿主，pET30a为表达载体，构建了鱼精蛋白的融合蛋白表达系统。IPTG诱导表达后，SDS-PAGE电泳显示实验组比对照组增加了一条具有预期分子量的亮带，初步证实了融合蛋白表达系统的成功构建。
Protamine is a kind of calionic antimicrobial pcptidc(CAP) found in the nuclei of sperm of different animals species, where it replaces histone-protamine during maturation of the spermatid. Studies indicate that, being a novel natural food preservative, protamine is effective, safe, with broad anti-microbial spectrum and worthy of further exploitation. Furthermore, protamine is beneficial to health because of its nutrition and functionality, it also has been applied broadly in medical treatment.
To provide some reference to the application of protamine, the experiment studied some physical and chemical factors which affect protamine's antimicrobial efficacy, in addition, we have cloned coding sequence of protamine by PCR, and expressed the fusion protein of protamine in E.coli. The main results were as following:
(1) Metal ions like Ca2+ Mg2+, K+% Na+ have negative effects on antimicrobial efficacy of protamine. The higher charge and concentration of metal ions, the bigger their effects on antimicrobial efficacy of protamine, and the degree hirarchy of effect was: Ca2+>Mg2+>K+>Na+.
(2) Antimicrobial efficacy of protamine is evidence under the condition of neutral and alkalescence, but it is low in the acidic condition.
(3) The temperature treatment show little effect on antimicrobial efficacy of protamine, which makes protamine ideal for application in food together with heat treatment to prolong shelf life of food.
(4) antimicrobial action of protamine is rapid and immediate effect, it can inhibit or kill majority of microorganism during a short time.
(!>) Organic substances such as protein, fat and sugar in food have a small effect on antimicrobial efficacy of protamine. among three organic substances, the effect of fat is more significant than other.
(6) Being a small peptide molecule, protamine's basic structure can be destroyed and it's antimicrobial efficacy largely affected by some protein enzymes like chymotrypsin, trypsin, papain and pepsin.
(7) A fragment of protamine DNA was cloned by PCR, and its sequence determined after cloning into a pUCT vector . Compared with known protamine gene sequence, there are three single nucleic acid variations in our senquence but their homology is up to 91.1%.
(8) we have also constructed the fusion expression system for prolamine using pET30a as expression vector and E.coli as expression strain. When induced by IPTG, the system expressed a new approximate 25 KD protain as checked by SDS-PAGE electrophoresis. we tentatively think that it is the target fusion protein of protamine combined with E.coli protein .
引文
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