中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2010年
4期
335-340
,共6页
林妙端%陈建森%佘菲菲%陈豪
林妙耑%陳建森%佘菲菲%陳豪
림묘단%진건삼%사비비%진호
幽门螺杆菌%oipA基因%核酸疫苗%优化%减毒沙门菌
幽門螺桿菌%oipA基因%覈痠疫苗%優化%減毒沙門菌
유문라간균%oipA기인%핵산역묘%우화%감독사문균
Helicobacter pylori(H.pylori)%oipA gene%DNA vaccine%Optimization%Attenuated s.typhimurium
目的:优化幽门螺杆菌(Helicobacter pylori,H.pylori)oipA核酸疫苗,构建含oipA核酸的SL7207(减毒伤寒沙门氏菌)重组菌株,并研究其在防御H.pylori感染中的作用.方法:对oipA基因进行密码子优化,Kozak前导序列添加、CpG序列优化,将oipA优化前后的基因分别克隆入pVAX1真核表达载体,获得pVAX1-oipA和pVAX1-optioipA重组子,将上述重组子与空载体pVAX1瞬时转染AGS细胞,应用Western blot检测转染细胞中OipA蛋白的表达.将重组子转化LB5000进行甲基化修饰,利用修饰后重组子转化终宿主菌SL7207,提取SL7207/pVAX1-oipA,SL7207/pVAX1-optioipA菌株质粒进行PCR,酶切鉴定.用SL7207/pVAX1-oipA和SL7207/pVAX1-optioipA免疫C57BL/6小鼠,末次免疫2周后检测抗OipA抗体,并在末次免疫4周后予H.pylori SS1(5 × 10~8CFU/只)攻击,攻击3次,隔天一次,攻击4周后处死动物,取胃组织做快速尿素酶实验和H.pylori定量培养以检测H.pylori感染状况.结果:pVAX1-oipA、pVAX1-optioipA均可在AGS细胞中表达,且pVAX1-optioipA的OiDA蛋白表达量高于pVAX1-oipA;从SL7207/pVAX1-oipA和SL7207/pVAX1-optioipA抽提的质粒,经PCR,酶切鉴定正确;免疫小鼠血清中产生抗OipA蛋白的特异性抗体IgG,且pVAX1-optioipA疫苗组诱导IgG水平明显高于pVAX1-oipA疫苗组(P＜0.01);pVAX1-oipA、pVAX1-optioipA疫苗组H.pylori感染率分别为60%(9/15)、26.67%(4/15),明显低于对照组100%(10/10)(P＜0.01).结论:成功构建携带pVAX1-oipA,pVAX1-optioipA的SL7207重组菌株,并验证其对C57BL/6小鼠H.pylori感染具有免疫预防作用,且优化oipn基因有助于提高免疫保护效果.
目的:優化幽門螺桿菌(Helicobacter pylori,H.pylori)oipA覈痠疫苗,構建含oipA覈痠的SL7207(減毒傷寒沙門氏菌)重組菌株,併研究其在防禦H.pylori感染中的作用.方法:對oipA基因進行密碼子優化,Kozak前導序列添加、CpG序列優化,將oipA優化前後的基因分彆剋隆入pVAX1真覈錶達載體,穫得pVAX1-oipA和pVAX1-optioipA重組子,將上述重組子與空載體pVAX1瞬時轉染AGS細胞,應用Western blot檢測轉染細胞中OipA蛋白的錶達.將重組子轉化LB5000進行甲基化脩飾,利用脩飾後重組子轉化終宿主菌SL7207,提取SL7207/pVAX1-oipA,SL7207/pVAX1-optioipA菌株質粒進行PCR,酶切鑒定.用SL7207/pVAX1-oipA和SL7207/pVAX1-optioipA免疫C57BL/6小鼠,末次免疫2週後檢測抗OipA抗體,併在末次免疫4週後予H.pylori SS1(5 × 10~8CFU/隻)攻擊,攻擊3次,隔天一次,攻擊4週後處死動物,取胃組織做快速尿素酶實驗和H.pylori定量培養以檢測H.pylori感染狀況.結果:pVAX1-oipA、pVAX1-optioipA均可在AGS細胞中錶達,且pVAX1-optioipA的OiDA蛋白錶達量高于pVAX1-oipA;從SL7207/pVAX1-oipA和SL7207/pVAX1-optioipA抽提的質粒,經PCR,酶切鑒定正確;免疫小鼠血清中產生抗OipA蛋白的特異性抗體IgG,且pVAX1-optioipA疫苗組誘導IgG水平明顯高于pVAX1-oipA疫苗組(P＜0.01);pVAX1-oipA、pVAX1-optioipA疫苗組H.pylori感染率分彆為60%(9/15)、26.67%(4/15),明顯低于對照組100%(10/10)(P＜0.01).結論:成功構建攜帶pVAX1-oipA,pVAX1-optioipA的SL7207重組菌株,併驗證其對C57BL/6小鼠H.pylori感染具有免疫預防作用,且優化oipn基因有助于提高免疫保護效果.
목적:우화유문라간균(Helicobacter pylori,H.pylori)oipA핵산역묘,구건함oipA핵산적SL7207(감독상한사문씨균)중조균주,병연구기재방어H.pylori감염중적작용.방법:대oipA기인진행밀마자우화,Kozak전도서렬첨가、CpG서렬우화,장oipA우화전후적기인분별극륭입pVAX1진핵표체재체,획득pVAX1-oipA화pVAX1-optioipA중조자,장상술중조자여공재체pVAX1순시전염AGS세포,응용Western blot검측전염세포중OipA단백적표체.장중조자전화LB5000진행갑기화수식,이용수식후중조자전화종숙주균SL7207,제취SL7207/pVAX1-oipA,SL7207/pVAX1-optioipA균주질립진행PCR,매절감정.용SL7207/pVAX1-oipA화SL7207/pVAX1-optioipA면역C57BL/6소서,말차면역2주후검측항OipA항체,병재말차면역4주후여H.pylori SS1(5 × 10~8CFU/지)공격,공격3차,격천일차,공격4주후처사동물,취위조직주쾌속뇨소매실험화H.pylori정량배양이검측H.pylori감염상황.결과:pVAX1-oipA、pVAX1-optioipA균가재AGS세포중표체,차pVAX1-optioipA적OiDA단백표체량고우pVAX1-oipA;종SL7207/pVAX1-oipA화SL7207/pVAX1-optioipA추제적질립,경PCR,매절감정정학;면역소서혈청중산생항OipA단백적특이성항체IgG,차pVAX1-optioipA역묘조유도IgG수평명현고우pVAX1-oipA역묘조(P＜0.01);pVAX1-oipA、pVAX1-optioipA역묘조H.pylori감염솔분별위60%(9/15)、26.67%(4/15),명현저우대조조100%(10/10)(P＜0.01).결론:성공구건휴대pVAX1-oipA,pVAX1-optioipA적SL7207중조균주,병험증기대C57BL/6소서H.pylori감염구유면역예방작용,차우화oipn기인유조우제고면역보호효과.
Objective: To optimize Helicobacter pylori(H.pylori)oipA gene and develop strain of vaccine in Attenuated Salmonella typhimurium SL7207 haxboring recombinant plasmid pVAX1-oipA and pVAX1-optioipA, then to study its protection against H.pylori in C57BL/6 mice.Methods: oipA gene was modified by codon optimization,inserting the Kozak sequence and engineering CpG motifs.The optioipA gene and oipA gene was inserted into the eukaryotic expressing vector pVAX1 respectively to develop recombinant plasmid pVAX1-optioipA and pVAX1-oipA.The AGS cells were transfected by pVAX1-oipA,pVAX1-optioipA and pVAX1 respectively.Then the OipA protein expression was confirmed by Western blot.pVAX1-oipA,and pVAX1-optioipA were converted to LB5000 for methylation and then the modified eukaryoticvector was electrotransfered to final host SL7207 respectively, SL7207/pVAX1-oipA, SL7207/pVAX1-optioipA was identified with PCR and enzyme digestion.Specific serum IgG antibody was detected by indirect ELISA after oral inoculation with vaccine strains.Mice were challenged with live Sydney strain(SS1)three times at 0,2,4 d(5 × 10~8CFU/each mouse).Fonr weeks after challenge, the mice were sacrificed.The colonization of H.pylori in gastric mucosa were detected by rapid urease test and quantitative culture of H.pylori.Results: The AGS cells transfected with pVAX1-oipA and pVAX1-optioipA had expressed corresponding production, moreover, the expression level of pVAX1-optioipA was higher than those of pVAX1-oipA,SL7207/pVAX1-oipA and SL7207/pVAX1-optioipA confirmed with PCR,enzyme digestion;Two weeks after last immunization,immunized mice.were induced to produce anti-OipA IgG antibodies, and the levels of anti-OipA IgG antibody in pVAX1-optioipA group was obviously higher than in pVAX1-oipA group(P＜0.01); After challenged with SS1, the infection rate of pVAX1-oipA and pVAX1-optioipA group was 60%(9/15)and 26.67%(4/15), which was significantly lower than 100%(10/10)in the control group(P＜0.01).Conclusion:Attenuated Salmonella typhimurium SL7207 containing pVAX1-oipA, pVAX1-optioipA is successfully constructed.It can protect mice from H pylori infection and optimizing oipA gene can enhance the protection efficiency.
