动物医学进展
動物醫學進展
동물의학진전
PROGRESS IN VETERINARY MEDICINE
2014年
12期
27-31
,共5页
徐全圆%刘建华%苏艳%加尔肯%王世民%冉多良
徐全圓%劉建華%囌豔%加爾肯%王世民%冉多良
서전원%류건화%소염%가이긍%왕세민%염다량
马腺疫链球菌%FNE基因%序列分析%原核表达
馬腺疫鏈毬菌%FNE基因%序列分析%原覈錶達
마선역련구균%FNE기인%서렬분석%원핵표체
Streptococcus equinus%FNE gene%sequence analysis%prokaryotic expression
采用 PCR 方法从马腺疫链球菌新疆分离株中扩增 FNE 基因片段,克隆至 pMD19-T 载体。利用分子生物学软件分析测序结果,将 FNE 截短基因亚克隆至原核表达载体 pET-30a 中,转化到大肠埃希菌(E.coli)BL21(DE3)感受态细胞,以 IPTG 诱导 FNE 重组蛋白的表达,用 SDS-PAGE 和 Western blot 法分析蛋白表达及其反应原性。序列分析显示,其与 GenBank 收录的马腺疫链球菌4047(登录号YP002747216)核苷酸序列和氨基酸序列同源性均为99%。采用 PCR 法扩增到675 bp 的 FNE 截短基因片段构建重组表达载体获得重组蛋白,经 SDS-PAGE 分析在46 ku 处出现明显条带,Western blot 分析显示具有反应原性。成功克隆和表达了马腺疫链球菌的 FNE 截短基因,为重组 FNE 蛋白亚单位疫苗的研制奠定了基础。
採用 PCR 方法從馬腺疫鏈毬菌新疆分離株中擴增 FNE 基因片段,剋隆至 pMD19-T 載體。利用分子生物學軟件分析測序結果,將 FNE 截短基因亞剋隆至原覈錶達載體 pET-30a 中,轉化到大腸埃希菌(E.coli)BL21(DE3)感受態細胞,以 IPTG 誘導 FNE 重組蛋白的錶達,用 SDS-PAGE 和 Western blot 法分析蛋白錶達及其反應原性。序列分析顯示,其與 GenBank 收錄的馬腺疫鏈毬菌4047(登錄號YP002747216)覈苷痠序列和氨基痠序列同源性均為99%。採用 PCR 法擴增到675 bp 的 FNE 截短基因片段構建重組錶達載體穫得重組蛋白,經 SDS-PAGE 分析在46 ku 處齣現明顯條帶,Western blot 分析顯示具有反應原性。成功剋隆和錶達瞭馬腺疫鏈毬菌的 FNE 截短基因,為重組 FNE 蛋白亞單位疫苗的研製奠定瞭基礎。
채용 PCR 방법종마선역련구균신강분리주중확증 FNE 기인편단,극륭지 pMD19-T 재체。이용분자생물학연건분석측서결과,장 FNE 절단기인아극륭지원핵표체재체 pET-30a 중,전화도대장애희균(E.coli)BL21(DE3)감수태세포,이 IPTG 유도 FNE 중조단백적표체,용 SDS-PAGE 화 Western blot 법분석단백표체급기반응원성。서렬분석현시,기여 GenBank 수록적마선역련구균4047(등록호YP002747216)핵감산서렬화안기산서렬동원성균위99%。채용 PCR 법확증도675 bp 적 FNE 절단기인편단구건중조표체재체획득중조단백,경 SDS-PAGE 분석재46 ku 처출현명현조대,Western blot 분석현시구유반응원성。성공극륭화표체료마선역련구균적 FNE 절단기인,위중조 FNE 단백아단위역묘적연제전정료기출。
A fragment of FNE gene was amplified from Streptococcus equinus isolated from Xinjiang by PCR and cloned into pMD19-T vector.After sequence analysis by molecular biology softwares,the trun-cated FNE gene was subcloned into the prokaryotic expression vector pET-30a and resulting plasmid was transformed into host BL21 (DE3)cells,in which a recombinant protein was expressed by inducing with IPTG.Then the expression and reaction of the recombinant proteins were detected by SDS-PAGE and Western blot.Results:Sequence analysis indicated that a 675 bp fragment of FNE gene was obtained by amplification by PCR,whose relative similarities in nucleotide and amino acids compared to the published sequences of Streptococcus equinus 4047(GenBank accession number YP002747216)all were 99%,respec-tively.After transformation of recombinant expression plasmid into host BL21 (DE3)cells,the SDS-PAGE analysis revealed that the expressed recombinant protein was 46 ku.Western blot showed its reac-tivity.It is concluded that the expression of truncated FNE protein lays foundation for the development of recombinant subunit vaccines against equine strangles.