CAJ | 학술논문

目的：制备人A组轮状病毒VP4结构蛋白N端的刺突蛋白VP8卵黄抗体IgY，并检测其生物学特征。方法以轮状病毒VP4基因的DNA为模板，采用PCR方法扩增VP8 DNA序列。将VP8片段克隆到原核表达载体 pET28a 上，形成重组质粒pET28a-VP8，转化基因工程菌E．coli BL21，完成VP8重组蛋白的诱导表达。 VP8包涵体蛋白经纯化和蛋白复性后，与弗氏佐剂混匀免疫产蛋鸡，收集鸡蛋，用水稀释-超滤法纯化卵黄抗体IgY。通过SDS-PAGE、Western blot、ELISA和点杂交等技术方法，分析该抗体的生物学特性，包括抗体的纯度、滴度、特异性和稳定性。结果 VP8在基因工程菌E．coli BL21中主要以包涵体蛋白形式表达，相对分子质量约为35×103，在pH8．0的Tris-HCl缓冲体系其复性效率可达90％。将重组抗原VP8免疫产蛋鸡，获得VP8抗原特异性的卵黄抗体IgY，抗体滴度达到1∶100000，且特异性地识别野生型A组轮状病毒。结论本研究采用基因工程和免疫技术制备的VP8卵黄抗体IgY，具有较高的抗体滴度和特异性，为婴幼儿轮状病毒感染性腹泻的诊断和防治提供了新的思路。
목적：제비인A조륜상병독VP4결구단백N단적자돌단백VP8란황항체IgY，병검측기생물학특정。방법이륜상병독VP4기인적DNA위모판，채용PCR방법확증VP8 DNA서렬。장VP8편단극륭도원핵표체재체 pET28a 상，형성중조질립pET28a-VP8，전화기인공정균E．coli BL21，완성VP8중조단백적유도표체。 VP8포함체단백경순화화단백복성후，여불씨좌제혼균면역산단계，수집계단，용수희석-초려법순화란황항체IgY。통과SDS-PAGE、Western blot、ELISA화점잡교등기술방법，분석해항체적생물학특성，포괄항체적순도、적도、특이성화은정성。결과 VP8재기인공정균E．coli BL21중주요이포함체단백형식표체，상대분자질량약위35×103，재pH8．0적Tris-HCl완충체계기복성효솔가체90％。장중조항원VP8면역산단계，획득VP8항원특이성적란황항체IgY，항체적도체도1∶100000，차특이성지식별야생형A조륜상병독。결론본연구채용기인공정화면역기술제비적VP8란황항체IgY，구유교고적항체적도화특이성，위영유인륜상병독감염성복사적진단화방치제공료신적사로。
Objective To prepare an egg yolk antibody ( IgY) against the recombinant human A rotavirus VP8 and evaluate its biological characteristics .Methods The complete VP4 gene was used as the template to clone VP8 DNA fragment by PCR .The VP8 gene was then cloned into the vector pET 28a for ex-pression of VP8 protein in the prokaryotic system.The expression plasmid of pET28a-VP8,identified by DNA sequencing,was transformed into E.coli BL21 to induce the expression of protein by 0.5 mmol/L IPTG at 30℃.The purified and refolded recombinant VP 8 protein was used to immunize laying hens in combination with complete Freund′s adjuvant ( CFA ) .Anti-VP8 IgY antibodies were extracted from egg yolks by using water dilution-ultrafiltration assay and were further analyzed by SDS-PAGE,ELISA,Western blot and Dot blot assay,respectively.Results The recombinant VP8 protein was expressed in E.coli BL21 as an inclusion body and its molecular weight was about 35 ×10 3 .The VP8 protein was well refolded in a buffer containing Tris-HCl (pH8.0).The isolated anti-VP8 IgY antibodies showed a high titer of 1∶100 000 and a high stabil-ity under the condition of pH3-11 and temperature 25-65℃.Moreover,the anti-VP8 IgY antibodies specific-ally recognized the wild type human A rotavirus .Conclusion The prepared anti-VP8 IgY antibodies showed the advantages of high titer ,good specificity and stability .It might be used as the tool in further investigation for the prevention and treatment of diarrhea caused by human A rotavirus .