重组乳酸乳球菌表达呼吸道合胞病毒Pre F的免疫原性分析  

Immunogenicity analysis of respiratory syncytial virus Pre F expressed by recombinant Lactococcus lactis

作　　者：赵同悦 高婧 袁蕾[2] 雷涵 ZHAO Tongyue;GAO Jing;YUAN Lei;LEI Han(Chengdu Medical College,Chengdu 610500,Sichuan Province,China;不详)

机构地区：[1]成都医学院,四川成都610500 [2]西南交通大学医学院,四川成都610031

出　　处：《中国生物制品学杂志》2024年第9期1037-1042,1049,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(32070920);中央高校基本科研业务费专项资金(2682023ZTPY06)。

摘　　要：目的 基于乳酸乳球菌(Lactococcus lactis,L.lactis)诱导表达系统制备呼吸道合胞病毒(respiratory syncytial virus,RSV)口服疫苗,并对其免疫原性进行分析。方法 将构建的质粒pUC57-Pre F与pNZ8149分别经NcoⅠ和KpnⅠ双酶切,酶切产物经T4 DNA连接酶连接后获得重组质粒pNZ8149-Pre F,电转化至感受态L.lactis NZ3900,经乳糖培养基筛选获得非分泌型重组L.lactis NZ3900/pNZ8149-Pre F,以nisin A为诱导剂诱导表达,经Western blot和免疫荧光标记对其表达产物进行分析;将雌性BALB/c小鼠随机分为PBS、L.lactis NZ3900/pNZ8149、L.lactis NZ3900/pNZ8149-Pre F组,15只/组,每只口服500μL,于第1、2天进行初次免疫,第16、17天进行加强免疫,第14和28天经小鼠下颌取血并分离脾脏细胞,ELSIA法检测重组L.lactis NZ3900/pNZ8149-Pre F诱导的体液和黏膜免疫应答水平,ELISpot法检测重组L.lactis NZ3900/pNZ8149-Pre F诱导的细胞免疫应答水平。结果 重组L.lactis NZ3900/pNZ8149-Pre F经PCR及测序鉴定,证明构建正确;抗原蛋白Pre F特异性地表达在L.lactis NZ3900中,相对分子质量约50 000;初次免疫后第14和28天,与PBS和L.lactis NZ3900/pNZ8149组相比,L.lactis NZ3900/pNZ8149-Pre F组小鼠血清Pre F-特异性IgG抗体效价、肠洗液中特异性分泌型IgA抗体以及脾脏细胞中IFNγ和IL-4分泌水平均明显升高,且差异均有统计学意义(t=-30.268~-3.087,P均<0.05)。结论 基于L.lactis诱导表达系统构建的RSV口服疫苗具有较强的免疫原性,为研发RSV黏膜疫苗提供了参考,也为开发其他病毒或细菌口服疫苗提供了新的研究策略及方法。Objective To prepare the oral vaccine of respiratory syncytial virus(RSV)based on Lactococcus lactis(L.lactis)induction expression system and analyze its immunogenicity.Methods The constructed plasmids pUC57-Pre F and pNZ8149 were digested with NcoⅠand KpnⅠ,respectively,and the digested products were ligated with T4 DNA ligase to obtain the recombinant plasmid pNZ8149-Pre F,which was electroporated into the competent L.lactis NZ3900,and then screened by lactose medium to obtain non-secretory recombinant L.lactis NZ3900/pNZ8149-Pre F. Using nisin A as the inducer,the expressed products were analyzed by Western blot and immunofluorescence labeling technique. Further,female BALB/c mice were randomly divided into PBS,L. lactis NZ3900/pNZ8149,and L. lactis NZ3900/pNZ8149-Pre F groups,with 15 mice in each group and oral administration 500 μL per mouse. The initial immunization was performed on the 1 d and 2 d,and the booster immunization was performed on the 16 d and 17 d. On the 14 d and 28 d,blood samples were taken from the mandible of mice and spleen cells were isolated. The levels of humoral and mucosal immune responses induced by recombinant L.lactis NZ3900/pNZ8149-Pre F were determined by ELISA,while the levels of cellular immune responses by ELISpot.Results The recombinant L.lactis NZ3900/pNZ8149-Pre F was constructed correctly as identified by PCR and sequencing. The antigen protein Pre F was specifically expressed in L.lactis NZ3900 with a relative molecular mass of about 50 000. Compared with PBS and L.lactis NZ3900/pNZ8149 groups,mice in L.lactis NZ3900/pNZ8149-Pre F group had significantly higher serum Pre F-specific IgG titers,specific secretory IgA antibody levels in intestinal lavage fluid,IFNγ and IL-4 secretion levels in spleen cells on the 14 d and 28 d after the initial immunization(t =-30. 268--3. 087,each P < 0. 05).Conclusion The RSV oral vaccine constructed based on L.lactis induction expression system has strong immunogenicity,which provides a reference for the development of RSV mucosal

关 键 词：乳酸乳球菌 诱导表达 呼吸道合胞病毒 口服疫苗 免疫原性 

分 类 号：R392[医药卫生—免疫学]