中国兽药杂志
中國獸藥雜誌
중국수약잡지
CHINESE JOURNAL OF VETERINARY DRUG
2012年
12期
9-12
,共4页
李公美%胡静涛%李玉梅%李茂辉%闫金明%刘可越%钱爱东
李公美%鬍靜濤%李玉梅%李茂輝%閆金明%劉可越%錢愛東
리공미%호정도%리옥매%리무휘%염금명%류가월%전애동
吉林白鹅%α干扰素%抗血清
吉林白鵝%α榦擾素%抗血清
길림백아%α간우소%항혈청
Jilin white goose%IFN -α%antisera
采用PCR方法扩增了吉林白鹅α干扰素(JL-GolFN—α)成熟肽编码序列,将IFN—α片段定向插入原核表达载体pGEX-6p-1中,构建重组质粒pGEX—IFN—α,将重组质粒转入大肠杆菌BL21(DE3)的感受态细胞里,在IPTG诱导下表达可溶性的融合蛋白(GST—IFN—α)。SDS—PAGE、Western—blot检测结果表明,重组吉林白鹅IFN—α融合蛋白(rJL—GoIFN-α)的分子量大小约为43ku,表达量占菌体总蛋白的25%。重组吉林自鹅仅干扰素,经谷胱甘肽Sepbarose-4B亲和柱层析纯化后,经过透析复性,得到纯化的目的蛋白,含量可达0.25mg/mL。将复性蛋白免疫新西兰白兔3次,制备高滴度的鹅IFN—α抗血清。本实验表达和纯化了鹅的IFN—α,并制备了兔抗鹅的IFN—α抗血清,为下一阶段鹅α干扰素重组蛋白的应用奠定了基础。
採用PCR方法擴增瞭吉林白鵝α榦擾素(JL-GolFN—α)成熟肽編碼序列,將IFN—α片段定嚮插入原覈錶達載體pGEX-6p-1中,構建重組質粒pGEX—IFN—α,將重組質粒轉入大腸桿菌BL21(DE3)的感受態細胞裏,在IPTG誘導下錶達可溶性的融閤蛋白(GST—IFN—α)。SDS—PAGE、Western—blot檢測結果錶明,重組吉林白鵝IFN—α融閤蛋白(rJL—GoIFN-α)的分子量大小約為43ku,錶達量佔菌體總蛋白的25%。重組吉林自鵝僅榦擾素,經穀胱甘肽Sepbarose-4B親和柱層析純化後,經過透析複性,得到純化的目的蛋白,含量可達0.25mg/mL。將複性蛋白免疫新西蘭白兔3次,製備高滴度的鵝IFN—α抗血清。本實驗錶達和純化瞭鵝的IFN—α,併製備瞭兔抗鵝的IFN—α抗血清,為下一階段鵝α榦擾素重組蛋白的應用奠定瞭基礎。
채용PCR방법확증료길림백아α간우소(JL-GolFN—α)성숙태편마서렬,장IFN—α편단정향삽입원핵표체재체pGEX-6p-1중,구건중조질립pGEX—IFN—α,장중조질립전입대장간균BL21(DE3)적감수태세포리,재IPTG유도하표체가용성적융합단백(GST—IFN—α)。SDS—PAGE、Western—blot검측결과표명,중조길림백아IFN—α융합단백(rJL—GoIFN-α)적분자량대소약위43ku,표체량점균체총단백적25%。중조길림자아부간우소,경곡광감태Sepbarose-4B친화주층석순화후,경과투석복성,득도순화적목적단백,함량가체0.25mg/mL。장복성단백면역신서란백토3차,제비고적도적아IFN—α항혈청。본실험표체화순화료아적IFN—α,병제비료토항아적IFN—α항혈청,위하일계단아α간우소중조단백적응용전정료기출。
The interferon a gene of Jilin white goose was amplified from genome DNA, the segment coding mature peptide of the GolFN -α gene was cloned into prokaryotic expression vector pGEX - 6p - 1 to construct recombinant plasmid pGEX - IFN -α. pGEX - IFN -α was transformed into E. coli BL - 21 ( DE3 ) , which can express fused protein GST - IFN -α in E. coli BL - 21 ( DE3 ) by IPTG inducing. The expressed product was identified by SDS - PAGE and Western blot. The results showed that fusion protein GST - IFN -α was about 45 ku and it could react with interferon antibody. After purificating with Glutathione Sepharose -4B affinity column, and then refolded by dialysis against water, antisera against goose IFN -a was generated by immunizing rabbis with the purified protein. The expression and purification of recombinant goose IFN - ct fusion protein and preparation of the antisera against goose IFN -α will lay a foundation for the future application of goose IFN -α.