生物技术进展
生物技術進展
생물기술진전
2013年
4期
252-256
,共5页
钟鲁龙%易咏竹%丁农%张金卫%柳纪省
鐘魯龍%易詠竹%丁農%張金衛%柳紀省
종로룡%역영죽%정농%장금위%류기성
猪干扰素%抗病毒活性%家蚕%杆状病毒%真核表达
豬榦擾素%抗病毒活性%傢蠶%桿狀病毒%真覈錶達
저간우소%항병독활성%가잠%간상병독%진핵표체
porcine interferon%antiviral activity%silkworm%baculovirus%eukaryotic expression
本研究利用家蚕杆状病毒表达系统在家蚕中表达猪α干扰素。首先优化并合成猪α干扰素基因，将其克隆到杆状病毒转移载体pVL1393的多角体基因启动子下游，与线性化的Bm-BacPAK6 DNA共转染至Bm-N细胞，在细胞内进行同源重组后，纯化得到重组病毒。 Western杂交结果显示，在蚕血淋巴中可检测到猪α干扰素表达。用微量细胞病变抑制法在Vero/VSV*GFP系统上进行干扰素活性测定，结果表明重组猪α干扰素有抗病毒活性，效价达到106 U/mL以上，证明在家蚕幼虫体内成功表达了有活性的猪α干扰素。
本研究利用傢蠶桿狀病毒錶達繫統在傢蠶中錶達豬α榦擾素。首先優化併閤成豬α榦擾素基因，將其剋隆到桿狀病毒轉移載體pVL1393的多角體基因啟動子下遊，與線性化的Bm-BacPAK6 DNA共轉染至Bm-N細胞，在細胞內進行同源重組後，純化得到重組病毒。 Western雜交結果顯示，在蠶血淋巴中可檢測到豬α榦擾素錶達。用微量細胞病變抑製法在Vero/VSV*GFP繫統上進行榦擾素活性測定，結果錶明重組豬α榦擾素有抗病毒活性，效價達到106 U/mL以上，證明在傢蠶幼蟲體內成功錶達瞭有活性的豬α榦擾素。
본연구이용가잠간상병독표체계통재가잠중표체저α간우소。수선우화병합성저α간우소기인，장기극륭도간상병독전이재체pVL1393적다각체기인계동자하유，여선성화적Bm-BacPAK6 DNA공전염지Bm-N세포，재세포내진행동원중조후，순화득도중조병독。 Western잡교결과현시，재잠혈림파중가검측도저α간우소표체。용미량세포병변억제법재Vero/VSV*GFP계통상진행간우소활성측정，결과표명중조저α간우소유항병독활성，효개체도106 U/mL이상，증명재가잠유충체내성공표체료유활성적저α간우소。
Porcine interferon-alpha ( PoIFNα) was expressed in silkworm larvae using silkworm-baculovirus expression system. The porcine interferon-alpha gene was artificially synthesized after codon optimization. The optimized target gene was cloned to the baculovirus transfer vector pVL1393 and the recombinant plasmid pVL-PoIFNα was constructed. pVL-PoIFNα was co-transfected with linearized Bm-BacPAK6 DNA into Bm-N cells. The recombinant proteins were successfully detected in hemolymph by Western blot analysis. The recombinant PoIFN-alpha expressed in hemolymph were verified to be of high antiviral activity by inhibiting the cytopathic effect of VSV*GFP in Vero cells, which was about 106 U/mL.
