CAJ | 학술논문

　　目的应用汉逊酵母表达系统进行肠道病毒71型（EV71）病毒样颗粒（VLP）的表达。方法将经过汉逊酵母密码子优化的人EV71的 P1和3CD基因片段克隆到汉逊酵母表达载体PMV上，获得重组表达质粒PMV-P1-3CD，转化汉逊酵母宿主菌AU0501，PCR方法及稳定传代培养筛选整合P1和3 CD基因的重组菌株。重组菌种接种在含有1％甲醇的培养基中进行诱导表达，对表达产物进行SDS-PAGE、Western blot检测。挑选优胜表达菌株进行30 L发酵罐发酵培养，发酵产物经过粗略纯化后进行电镜分析。结果筛选获得EV71重组表达菌株；SDS-PAGE检测结果显示在相对分子质量（Mr）为26×103、33×103、35×103处有明显的VP3、VP1、VP0蛋白条带，Mr 大小与预期的目的蛋白大小一致；Western blot检测结果显示表达产物与EV71-VP1单克隆抗体在M r 为33×103处有较为明显的VP1反应条带，表达产物具有良好的免疫反应性；表达菌株发酵表达量可达200 mg/L，电镜分析可见24～30 nm的VLP，且颗粒结构完好。结论应用汉逊酵母表达系统成功表达了EV71 VLP，为今后研制EV71 VLP疫苗奠定基础。
　　목적응용한손효모표체계통진행장도병독71형（EV71）병독양과립（VLP）적표체。방법장경과한손효모밀마자우화적인EV71적 P1화3CD기인편단극륭도한손효모표체재체PMV상，획득중조표체질립PMV-P1-3CD，전화한손효모숙주균AU0501，PCR방법급은정전대배양사선정합P1화3 CD기인적중조균주。중조균충접충재함유1％갑순적배양기중진행유도표체，대표체산물진행SDS-PAGE、Western blot검측。도선우성표체균주진행30 L발효관발효배양，발효산물경과조략순화후진행전경분석。결과사선획득EV71중조표체균주；SDS-PAGE검측결과현시재상대분자질량（Mr）위26×103、33×103、35×103처유명현적VP3、VP1、VP0단백조대，Mr 대소여예기적목적단백대소일치；Western blot검측결과현시표체산물여EV71-VP1단극륭항체재M r 위33×103처유교위명현적VP1반응조대，표체산물구유량호적면역반응성；표체균주발효표체량가체200 mg/L，전경분석가견24～30 nm적VLP，차과립결구완호。결론응용한손효모표체계통성공표체료EV71 VLP，위금후연제EV71 VLP역묘전정기출。
Objective To express virus-like particles(VLP) of enterovirus 71 (EV71) in Han-senula polymorpha.Methods The coding sequences of P1 and 3CD genes of EV71 were optimized accord-ing to codon usage bias of Hansenula polymorpha for achieving high expression , and then cloned into the ex-pression vector PMV of Hansenula polymorpha .The recombinant expression vector PMV-P1-3CD was trans-formed into Hansenula polymorpha AU 0501 .The transformants were stably cultured in selective medium (Yeast Nitrogen Base) and screened for strains with positive P1 and 3CD genes by PCR.Then an induced cultivation on the recombinant strains were performed in a medium supplemented with methanol to a final concentration of 1.0%and the expressed products were analyzed by SDS-PAGE and Western blot assays to select high expression strains .The high expression strains were cultured in 30 L fermentor and its fermenta-tion products were analyzed by electronic microscope after purification .Results EV71 recombinant expres-sion strains were successfully constructed .The results of SDS-PAGE showed that the expressed products had obvious VP3, VP1, VP0 protein bands with molecular weights of 26×103, 33×103 and 35×103, respective-ly, which were consistent with the expected molecular weight of the fusion proteins .Western blot demonstra-ted that the expressed products could be specifically recognized by the polyclonal antibody against EV 71-VP1 at 33 ×10 3 , indicating its high immunoreactivity .ELISA confirmed that the expression level of EV 71 fermen-tation products was reached to 200 mg/L.Electronic microscope analysis showed that the VLP of recombi-nant EV71 were 24-30 nm in diameter with normal structure .Conclusion The virus-like particles of human enterovirus 71 are successfully expressed in Hansenula polymorpha , which provides a foundation for the fur-ther development of EV 71 VLP vaccine .