摘要
为表达鸡胸腺素α1(Thymosin alpha l,Tα1),并对其进行纯化及生物学活性分析,本研究根据大肠杆菌基因密码子偏嗜性对Tα1基因进行优化,人工合成了Tα1三拷贝融合基因。并将其克隆至原核表达载体pET-30a中,转化至BL21(DE3)菌株,经IPTG诱导表达和镍柱纯化,获得融合蛋白。融合蛋白经羟胺切割后获得Tαl单体。将单体Tα1按高、中、低3个剂量肌肉注射160只7日龄非免疫商品肉鸡,于不同日龄比较Tα1单体对鸡体内淋巴细胞刺激指数(SI)的影响。结果显示,鸡Tα1三串体为约19 ku大小的融合蛋白,单体约为3.0 ku,与天然Tα1相符。活性分析结果显示,0.03 mg/kg、0.06 mg/kg和0.12 mg/kg 3个剂量的Tα1均能提高鸡体内SI,其中21日龄、28日龄、35日龄各试验组与对照组相比均表现差异显著(p<0.05),以高剂量组最为明显,各试验组间差异不显著。研究表明,通过表达串联体基因方式制备有活性的Tα1是可行的,为Tα1进一步在兽医临床应用提供依据。
To express thymosin alpha 1（Tα1）,and identify its bioactivity,a codon optimized triple-copy fusion gene of chicken Tα1 was chemically systhesized according to the published sequence with hydroxylamine schizolysis site between each copy.The fusion gene was cloned into pET-30a and transformed into BL21（DE3） for expression.The fusion protein was expressed with IPTG induction and purified by NI-NTA.The Tα1 monomer was prepared by cleavaged with hydroxylamine.A total of 160 chickens was divided into 4 groups and injected at high,middle and low dose,respectively,to detect the lymphocyte stimulating index（SI） in different period.The results showed that the fusion protein was 19 ku,monomer was 3.1 ku.The difference of SI were significant between experiment groups and control group（p0.05） and the high dose group was better than that of other groups.Therefore,it is a feasible method in perparing micromolecule polypeptide by fusion expression.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2011年第2期150-153,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
青岛城阳区科技发展计划课题(2006-NY-4)
关键词
鸡胸腺素
大肠杆菌
融合表达
纯化
活性分析
chicken thymosin
Escherichia coli
fusion expression
purification
activity analysis
