CAJ | 학술논문

目的构建嵌合preS1 21-47位氨基酸(aa)、以全长乙肝病毒(HBV)核心抗原(HBcAg)为骨架的病毒样颗粒H2和嵌合preS1 aa 37-45、以截断HBcAg为骨架的病毒样颗粒H3,并对比其抗原性.方法将HBVadr血清型preS1中和表位aa 21-47和aa 37-45分别插入到全长HBc(aa 1-183)和截断HBc (aa 1-144)的第78位天冬氨酸和第79位脯氨酸之间,密码子优化之后全基因合成,目的片段经双酶切(NdeI和XhoI)回收后连接到同样处理的pET43.1a载体上,在大肠埃希菌E.coli BL21(DE3)中表达,精细纯化后通过电镜观察蛋白形态、ELISA以及Western Blott检测其抗原性.结果成功构建表达质粒H2和H3,并在BL21(DE3)中高效表达和自发组装成病毒样颗粒.纯化后电镜观察H2和H3形态为二十面体立体对称样病毒样颗粒,H2为实心颗粒,直径为(31.61±1.27) nm,H3为空心颗粒,直径为(28.46 ±1.16) nm.统计分析可得H2直径要比H3直径大(P＜0.01).ELISA和Western Blott检测结果证实其插入表位preS1以及载体蛋白HBc的抗原性.结论成功获得两种嵌合HBV preS1中和表位的病毒样颗粒,为其在疫苗研究中的应用探索奠定基础.
목적 구건감합preS1 21-47위안기산(aa)、이전장을간병독(HBV)핵심항원(HBcAg)위골가적병독양과립H2화감합preS1 aa 37-45、이절단HBcAg위골가적병독양과립H3,병대비기항원성.방법 장HBVadr혈청형preS1중화표위aa 21-47화aa 37-45분별삽입도전장HBc(aa 1-183)화절단HBc (aa 1-144)적제78위천동안산화제79위포안산지간,밀마자우화지후전기인합성,목적편단경쌍매절(NdeI화XhoI)회수후련접도동양처리적pET43.1a재체상,재대장애희균E.coli BL21(DE3)중표체,정세순화후통과전경관찰단백형태、ELISA이급Western Blott검측기항원성.결과 성공구건표체질립H2화H3,병재BL21(DE3)중고효표체화자발조장성병독양과립.순화후전경관찰H2화H3형태위이십면체입체대칭양병독양과립,H2위실심과립,직경위(31.61±1.27) nm,H3위공심과립,직경위(28.46 ±1.16) nm.통계분석가득H2직경요비H3직경대(P＜0.01).ELISA화Western Blott검측결과증실기삽입표위preS1이급재체단백HBc적항원성.결론 성공획득량충감합HBV preS1중화표위적병독양과립,위기재역묘연구중적응용탐색전정기출.
Objective To construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity.Methods PreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg(aa 1-144),between the 78th (Asp) and 79th (Pro).The genes synthesized after the codon optimization were ligated to the pET43.1 a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E.coli expression system.The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting.Results The morphology of the virus-like particles were confirmed by electron microscope.H2 were solid particles with a diameter of (31.61 ± 1.27) nm,while H3 were hollow particles with a diameter of (28.46 ± 1.16) nm.Statistical analysis showed that H2 is larger than H3 in the diameter (P ＜ 0.01).The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting.Conclusion Chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed,purified and identified,which lays the foundation for its application in vaccine research.