摘要
根据GenBank中鸡IFN—α序列设计引物对，利用PCR技术克隆并测定了莱航鸡（SPY）的IFN-α基因，命名ChIFN-S1，与GenBank中IFN-α比较，ORF长度均为582个核苷酸，其成熟肽包含162个氨基酸，相互间同源性在98．8％-99．8％。将其成熟肽基因序列克隆入pQE30表达载体后，通过ITPG诱导，收集菌体高压破碎后提取包涵体并进行初步纯化，利用胱氨酸．半胱氨酸再氧化还原法对纯化后的变性液进行分段稀释法复性。细胞病变抑制法（CEF-VSV为检测系统）测定复性液的抗病毒比活性高达10^9U／mg以上．
In order to obtain high chlFN-α mature protein gene was reaction （PCR）, and was cloned expression of Chicken Interferon-alpha （chlFN-α） in Escherichia coli, the obtained from genomic DNA of layer （ Leghorn chicken） by polymerase chain into pMD18-T vector for sequencing. Sequencing results show that the open reading fragment （ORF） of the cloned chlFN-α gene consists of 582 nucleotides and the mature protein gene is composed of 486 nucleotides which encoding 162 amino acid, the homology with the chlFN-α gene from GenBank were 98.8% -99.8%. Then the mature protein gene was inserted into the SphI and Hind III site of the expression vector pQE30 and highly expressed in E. coli M15 which were further induced by IPTG and cultured, and a fusion protein was obtained with about 21ku of molecular weight in SDS-PAGE. The expressed chlFN-α account for 17.6% of the total cellular proteins and existed as inclusion body. The ehlFN-αinclusion body was dissolved in 7mol/L guanidine chloride contained 10 mmol/L DTT and subsequently the denatured chlFN-α was renatured by dilution in refolding buffer containing cystine and cysteine. In this study it was found that the denatured chlFN-α was efficiently renatured in refolding buffer containing 1 mol/L guanidine chloride and 0.5 mol/L arginine. As a result, the purified chIFN-α is verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in CEF cells, which is about 109 U/rag. This study paved the way for large-scale production of recombinant chIFN-αand its usage in virus disease control of chickens.
出处
《中国兽药杂志》
2007年第6期4-8,共5页
Chinese Journal of Veterinary Drug
关键词
鸡
IFN-α基因
表达
包涵体
复性
chicken interferon-α
gene expression
inclusion body
refolding
