人金属硫蛋白在乳酸菌中的融合表达及其预防食源性镉污染的应用 <sup>*</sup>

doi:10.13523/j.cb.20180306

中国生物工程杂志

2018, Vol. 38

Issue (3): 41-50 DOI: 10.13523/j.cb.20180306

技术与方法

人金属硫蛋白在乳酸菌中的融合表达及其预防食源性镉污染的应用 ^*

彭德莲¹,刘霞¹,黄亚东^1,²,肖雪^1*(

),苏志坚¹

1 暨南大学生命科学技术学院细胞生物学系广州 510632
2 广东省生物工程药物重点实验室广州 510632

Prevention of Gastrointestinal Cadmium Poisoning with Food-grade Lactococcus lactis Strain Expressing Metallothionein Fusion Protein

De-lian PENG¹,Xia LIU¹,Ya-dong HUANG^1,²,Xue XIAO^1*(

),Zhi-jian SU¹

1 Department of Cell Biology, College of Life Science and Technology, Jinan University, Guangzhou 510632,China
2 Guangdong Provincial Key Laboratory of Bioengineering Medicine, Institute of Biomedicine, Jinan University, Guangzhou　510632,China

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摘要： 目的

镉(Cd)是一种重要的环境污染物,其具有半衰期长、不能生物降解等特性使之易于在人体重要脏器中蓄积并对人身体健康造成不可逆的损伤。为此,减少镉的摄入,尤其是经胃肠道,是预防人体慢性镉中毒的有效方法。

方法

构建以α-半乳糖苷酶作为筛选标记、表达金属硫蛋白融合蛋白(GST-SUMO-MT)的重组乳酸乳球菌MG1363/pM-GSMT。原子吸收光谱法分析重组乳酸菌对Cd ²⁺和Zn ⁺的结合能力。以灌胃方式,对雄性Sprague-Dawley大鼠每天口服不同剂量的重组乳酸菌(2×10 ⁹~2×10 ¹¹ CFU/d)及CdCl2溶液[5mg/(kg·d)],连续处理56天。收集实验动物肝、肾、脑及睾丸,利用原子吸收光谱法检测这些脏器中镉离子的含量。生化分析检测血清中尿素及丙氨酸转氨酶的含量。石蜡切片及苏木精-伊红染色分析各组织的病理变化。

结果

获得不含抗生素抗性的重组乳酸菌MG1363/pM-GSMT,其吸附Cd ²⁺、Zn ²⁺能力比对照组分别提高3.52倍和1.60倍。动物实验结果显示,镉能在肝、肾、脑及睾丸中蓄积,并对这些器官造成明显的病理损伤。原子吸收光谱分析、血清生化指标及病理切片结果都显示,重组乳酸菌能有效降低胃肠道中镉的摄入,进而减少Cd ²⁺在各脏器中的蓄积及对器官功能的损伤。

结论

为预防人体食源性慢性镉中毒提供了一种有效的生物材料和使用方法。

关键词： 重组乳酸菌; 金属硫蛋白; 镉污染; 预防

Abstract: Objective:

Cadmium is an extremely toxic pullutant. The characterists of having long half-life and resistance to environmental degradation make it easy to accumulate in human organs and then cause irreversible damage. Blocking cadmium uptake via the gastrointestinal tract is an important prevention strategy.

Methods:

the recombinant Lactococcus lactis strain MG1363/pM-GSMT were constructed, which expressed fusion protein glutathione S-transferase(GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT) and using α- galactosidase as marker. The ability of binding cadmium or zinc ions was detected by the Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). For the animal experiments, the male Sprague-Dawley rats were given a daily oral administration of cadmium chloride solution [5mg/(kg·d)] and MG1363/pM-GSMT with different dosages respectively. This experiment was performed for 8 weeks and the tissues (liver, kidney, brain and testis) were collected. The accumulation of cadmium in tissues was determined by atomic absorption spectrometric analysis. The concentrations of alanine aminotransferase (ALT) and blood uria nitrogen (BUN) were analyzed by a full automated hematology analyzer. The pathological changes of each tissue treated by cadmium with/without MG1363/pM-GSMT were compared and analyzed using hematoxylin - eosin staining.

Results:

The recombinant strain MG1363/pM-GSMT with α-galactosidase resistant marker was constructed, which binding capacity of cadmium and zinc ions were 3.52 and 1.60 fold higher than that of control strain MG1363/pMG36e. After daily CdCl₂ treatment for 8 weeks, the results showed that the cadmium concentration increased significantly in the kidney, liver, brain and testis of experimental group compared with the normal group. The accumulated cadmium impaired the functions of liver and kidney. Oral administration of MG1363/pM-GSMT to SD rats that were also treated with cadmium chloride daily significantly blocked the cadmium uptake via gastrointestinal tract and further decreased the impairments of main tissues in morphology and functions.

Conclusion:

A potential biomaterial and application for preventing cadmium poisoning via the digestive tract.

Key words: Lactococcus lactis Metallothionein Cadmium poisoning Prevention

收稿日期: 2017-09-15 出版日期: 2018-04-04

ZTFLH:

Q819

基金资助: * 广东省高等学校珠江学者岗位计划(2012~2017年度)、广东省海洋经济创新发展区域示范专项(GD2012-B01-005)资助项目

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	彭德莲
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引用本文:

彭德莲, 刘霞, 黄亚东, 肖雪, 苏志坚. 人金属硫蛋白在乳酸菌中的融合表达及其预防食源性镉污染的应用 ^*[J]. 中国生物工程杂志, 2018, 38(3): 41-50.

De-lian PENG, Xia LIU, Ya-dong HUANG, Xue XIAO, Zhi-jian SU. Prevention of Gastrointestinal Cadmium Poisoning with Food-grade Lactococcus lactis Strain Expressing Metallothionein Fusion Protein. China Biotechnology, 2018, 38(3): 41-50.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180306 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I3/41

表1 重组质粒构建PCR引物表

图1 重组质粒pM-GSMT构建流程图

图2 菌落PCR方法扩增重组质粒中melA基因

图3 菌落PCR方法扩增重组质粒中编码GSMT基因

图4 MBCP平板检测重组质粒α-半乳糖苷酶活性

图5 重组乳酸菌株对红霉素抗性分析

图6 SDS-PAGE电泳检测纯化后的融合蛋白GSMT

图7 重组融合蛋白的Western blot分析

图8 重组乳酸菌对重金属离子吸附能力的分析

图9 不同处理组动物饲养过程中体重的变化分析

图10 不同处理组大鼠肾、肝、脑、睾丸组织中Cd2+的含量

图11 不同处理组大鼠血清中丙氨酸转移酶及尿素含量分析

图12 不同处理组大鼠肾脏组织病理图(HE染色)

图13 不同处理组大鼠肝脏组织病理图(HE染色)

图14 不同处理组大鼠睾丸组织病理图(HE染色)

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