中华地方病学杂志
中華地方病學雜誌
중화지방병학잡지
Chinese Journal of Endemiology
2013年
3期
263-266
,共4页
范金明%王发兴%张波%江玲%李蓓
範金明%王髮興%張波%江玲%李蓓
범금명%왕발흥%장파%강령%리배
布鲁杆菌%Ⅳ型分泌系统%免疫原性%疫苗
佈魯桿菌%Ⅳ型分泌繫統%免疫原性%疫苗
포로간균%Ⅳ형분비계통%면역원성%역묘
Brucella%Type Ⅳ secretion system%Immunogenicity%Vaccine
目的 检测布鲁杆菌Ⅳ型分泌系统蛋白VirB9的免疫原性,寻找潜在的、新的布鲁杆菌亚单位疫苗靶标.方法 采用双酶切的方法,将布鲁埃希菌VirB9基因全长克隆至原核表达载体pET32a上,将克隆后载有VirB9基因的pET32a质粒转化至大肠杆菌BL21 (DE3)中,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导重组VirB9蛋白在大肠埃希菌中的表达.利用重组VirB9蛋白所携带的His标签,通过Ni-NAT层析,纯化在大肠杆菌中重组表达的VirB9蛋白,再通过十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳和二喹啉甲酸(BCA)蛋白定量试剂盒对纯化的蛋白进行纯度及浓度检测.用布鲁杆菌疫苗株S19株免疫BAL B/c小鼠,并以磷酸盐缓冲液(PBS)为对照.在免疫4周后,鼠尾取血,血清虎红平板凝集试验、试管凝集试验检查小鼠血清抗体；酶联免疫吸附试验(ELISA)检测S19株免疫小鼠体内抗VirB9抗体滴度.在免疫后第35天,取小鼠脾脏,分离脾细胞,利用Elispot技术,检测VirB9蛋白体外再刺激后分泌细胞因子(干扰素-γ,IFN-γ)的脾细胞数,酶联斑点图像仪计数斑点,每个斑点代表1个抗原特异性T淋巴细胞,分析VirB9刺激机体产生细胞免疫反应的情况.结果 通过酶切克隆的方法成功地将VirB9全长基因741 bp克隆至pET32a载体上.SDS-PAGE显示,VirB9蛋白的相对分子质量约43×103,纯度超过97％；BCA法检测,蛋白浓度为1.6 g/L.免疫组小鼠血清虎红平板凝集试验阳性,试管凝集试验小鼠血清抗体滴度＞ 1∶800;对照组小鼠血清虎红平板凝集试验阴性,试管凝集试验小鼠血清抗体滴度阴性.免疫组检测到抗VirB9蛋白抗体,抗体滴度均＞1:3200,对照组小鼠体内未检测到抗VirB9蛋白抗体的存在.酶联斑点图像分析显示,用VirB9蛋白体外刺激,免疫组小鼠5×105个脾细胞中有147个细胞可以分泌IFN-γ,对照组仅有38个细胞.结论 在布鲁杆菌感染过程中,Ⅳ型分泌系统蛋白VirB9具有免疫原性,能够刺激小鼠产生体液免疫反应与细胞免疫反应.
目的 檢測佈魯桿菌Ⅳ型分泌繫統蛋白VirB9的免疫原性,尋找潛在的、新的佈魯桿菌亞單位疫苗靶標.方法 採用雙酶切的方法,將佈魯埃希菌VirB9基因全長剋隆至原覈錶達載體pET32a上,將剋隆後載有VirB9基因的pET32a質粒轉化至大腸桿菌BL21 (DE3)中,異丙基-β-D-硫代吡喃半乳糖苷(IPTG)誘導重組VirB9蛋白在大腸埃希菌中的錶達.利用重組VirB9蛋白所攜帶的His標籤,通過Ni-NAT層析,純化在大腸桿菌中重組錶達的VirB9蛋白,再通過十二烷基硫痠鈉-聚丙烯酰胺凝膠(SDS-PAGE)電泳和二喹啉甲痠(BCA)蛋白定量試劑盒對純化的蛋白進行純度及濃度檢測.用佈魯桿菌疫苗株S19株免疫BAL B/c小鼠,併以燐痠鹽緩遲液(PBS)為對照.在免疫4週後,鼠尾取血,血清虎紅平闆凝集試驗、試管凝集試驗檢查小鼠血清抗體；酶聯免疫吸附試驗(ELISA)檢測S19株免疫小鼠體內抗VirB9抗體滴度.在免疫後第35天,取小鼠脾髒,分離脾細胞,利用Elispot技術,檢測VirB9蛋白體外再刺激後分泌細胞因子(榦擾素-γ,IFN-γ)的脾細胞數,酶聯斑點圖像儀計數斑點,每箇斑點代錶1箇抗原特異性T淋巴細胞,分析VirB9刺激機體產生細胞免疫反應的情況.結果 通過酶切剋隆的方法成功地將VirB9全長基因741 bp剋隆至pET32a載體上.SDS-PAGE顯示,VirB9蛋白的相對分子質量約43×103,純度超過97％；BCA法檢測,蛋白濃度為1.6 g/L.免疫組小鼠血清虎紅平闆凝集試驗暘性,試管凝集試驗小鼠血清抗體滴度＞ 1∶800;對照組小鼠血清虎紅平闆凝集試驗陰性,試管凝集試驗小鼠血清抗體滴度陰性.免疫組檢測到抗VirB9蛋白抗體,抗體滴度均＞1:3200,對照組小鼠體內未檢測到抗VirB9蛋白抗體的存在.酶聯斑點圖像分析顯示,用VirB9蛋白體外刺激,免疫組小鼠5×105箇脾細胞中有147箇細胞可以分泌IFN-γ,對照組僅有38箇細胞.結論 在佈魯桿菌感染過程中,Ⅳ型分泌繫統蛋白VirB9具有免疫原性,能夠刺激小鼠產生體液免疫反應與細胞免疫反應.
목적 검측포로간균Ⅳ형분비계통단백VirB9적면역원성,심조잠재적、신적포로간균아단위역묘파표.방법 채용쌍매절적방법,장포로애희균VirB9기인전장극륭지원핵표체재체pET32a상,장극륭후재유VirB9기인적pET32a질립전화지대장간균BL21 (DE3)중,이병기-β-D-류대필남반유당감(IPTG)유도중조VirB9단백재대장애희균중적표체.이용중조VirB9단백소휴대적His표첨,통과Ni-NAT층석,순화재대장간균중중조표체적VirB9단백,재통과십이완기류산납-취병희선알응효(SDS-PAGE)전영화이규람갑산(BCA)단백정량시제합대순화적단백진행순도급농도검측.용포로간균역묘주S19주면역BAL B/c소서,병이린산염완충액(PBS)위대조.재면역4주후,서미취혈,혈청호홍평판응집시험、시관응집시험검사소서혈청항체；매련면역흡부시험(ELISA)검측S19주면역소서체내항VirB9항체적도.재면역후제35천,취소서비장,분리비세포,이용Elispot기술,검측VirB9단백체외재자격후분비세포인자(간우소-γ,IFN-γ)적비세포수,매련반점도상의계수반점,매개반점대표1개항원특이성T림파세포,분석VirB9자격궤체산생세포면역반응적정황.결과 통과매절극륭적방법성공지장VirB9전장기인741 bp극륭지pET32a재체상.SDS-PAGE현시,VirB9단백적상대분자질량약43×103,순도초과97％；BCA법검측,단백농도위1.6 g/L.면역조소서혈청호홍평판응집시험양성,시관응집시험소서혈청항체적도＞ 1∶800;대조조소서혈청호홍평판응집시험음성,시관응집시험소서혈청항체적도음성.면역조검측도항VirB9단백항체,항체적도균＞1:3200,대조조소서체내미검측도항VirB9단백항체적존재.매련반점도상분석현시,용VirB9단백체외자격,면역조소서5×105개비세포중유147개세포가이분비IFN-γ,대조조부유38개세포.결론 재포로간균감염과정중,Ⅳ형분비계통단백VirB9구유면역원성,능구자격소서산생체액면역반응여세포면역반응.
Objective To detect the immunogenicity of VirB9,a protein of type Ⅳ secretion system of Brucella.Methods Full length VirB9 gene was cloned into plasmid pET32a and expressed in Escherichia (E.) coli BL21 (DE3).Expression of recombinant protein was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) and the recombinant fusion protein was purified by affinity chromatography on Ni2+-conjugated chelateing sepharose.The purity of the purified protein was ascertained by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE) and the concentration was measured by bicinchoninic acid (BCA) protein assay kit.Animal model was established by immunizing BAL B/c mice with live vaccine strain S19 of Brucella and the mice immunized with phosphate buffered saline (PBS) as control.The blood of immunized mice was acquired after 4 weeks.Antibody against VirB9 in S19 immunized mice was detected by Rose Bengal plate agglutination test and serum tube agglutination test; IgG antibody titers against VirB9 in immunized mice were determined by enzyme linked immunosorbent assay(ELISA).At the 35th day,the immunized mice and control mice were killed and spleens were collected.The splenocytes were harvested and stimulated with each of VirB9,concanvalin A(ConA) or medium in triplicate.Production of gamma interferon (IFN-γ) was determined by enzyme-linked immunospot assay (Elispot).Results The full length of VirB9 gene was cloned into pET32a.The recombinant VirB9 protein was expressed at 43 × 103 in relative molecular mass and the purity of the purified recombinant VirB9 protein was above 97％ in SDS-PAGE and the concentration was 1.6 g/L in BCA protein assay.The antibody of VirB9 was detected in all S19 immunized mice but not PBS immunized mice by Rose Bengal plate agglutination test.The antibody titer in all S19 immunized mice was ＞ 1 ∶ 800 or ＞ 1 ∶ 3200 by tube agglutination test and ELISA,respectively.Meanwhile,the protein stimulated stronger IFN-γresponse in immunized mice than that in the control mice(147 cells Vs 38 cells).Conclusion VirB9 can stimulate humoral and cellular immunity and it might be an appropriate target for developing subunit vaccine against Brucella.
