CAJ | 학술논문

为了获得具有抗禽源病毒活性的鸡α干扰素，对Genebank中发表的鸡源α干扰素基因进行密码子优化后，人工合成α干扰素基因片段489 bp，构建了表达载体PET-28a-IFN-α，连接转化到大肠杆菌菌株中，经PCR、酶切和测序鉴定后，诱导产生干扰素包涵体蛋白，破碎、纯化、透析复性、冻干浓缩后测定蛋白浓度，按禽流感H9 EID50的浓度分别与不同剂量干扰素等量混合，接种鸡胚0.2μL，收集病毒，测定血凝价。结果显示：成功构建了原核表达载体pET-IFN-α，复性后蛋白浓度为472μg/mL，试验组（47.2μg和23.6μg）的血凝价与生理盐水对照组比较，差异极显著（P＜0.01）。表明重组干扰素蛋白在鸡胚内具有较好的抑制或杀灭H9禽流感病毒的效果，为下一步抗病毒制品的研发奠定了基础。
위료획득구유항금원병독활성적계α간우소，대Genebank중발표적계원α간우소기인진행밀마자우화후，인공합성α간우소기인편단489 bp，구건료표체재체PET-28a-IFN-α，련접전화도대장간균균주중，경PCR、매절화측서감정후，유도산생간우소포함체단백，파쇄、순화、투석복성、동간농축후측정단백농도，안금류감H9 EID50적농도분별여불동제량간우소등량혼합，접충계배0.2μL，수집병독，측정혈응개。결과현시：성공구건료원핵표체재체pET-IFN-α，복성후단백농도위472μg/mL，시험조（47.2μg화23.6μg）적혈응개여생리염수대조조비교，차이겁현저（P＜0.01）。표명중조간우소단백재계배내구유교호적억제혹살멸H9금류감병독적효과，위하일보항병독제품적연발전정료기출。
In order to obtain chicken interferon alpha (ChIFN-α) with the activity of inhibiting and killing poultry-origin virus,the 489 bp ChIFN-αgene from Genebank was designed and synthesized according to synonymous codon bias of E.coli,and GC-content was optimized.The synthetic ChIFN-α gene was inserted into a pro- karyotic expression vector pET-28a,and integrated into the genome of E.coli Rosetta strain through electroporation.The positive strain with the cor-rect reading frame was confirmed by PCR,enzyme digestion and sequencing.The protein in the form of inclusion body was expressed after induction,with its concentration measured following the whole process including ultrasonication,purifica-tion,dialysis-renaturation,and lyophilization.According to the concentration of H9 AIV EID50,the virus solution was equal-ly blended with ChIFN-α with different concentrations,and 0.2 μL mixture was injected into one chick embryo.After H9 AIV virus was collected from all the chick embryos for the antivirus experiment,hemagglutination valence was detected. The results indicated that the recombinant ChIFN-α protein was induced by IPTG with the concentration of 472 μg/mL. The groups treated with the protein of 47.2 μg and 23.6 μg revealed higher level of hemagglutination valence compared with control group( P<0.01),which proved that the recombinant ChIFN-αprotein had antiviral activity against H9 AIV. And it could promote the development of the antiviral products.