羊口疮病毒B2L截短重组蛋白刺激小鼠产生特异性抗体的研究

Study on specific antibody production stimulated by B2L truncated recombinant protein of orf virus

为研究羊口疮病毒(ORFV)新型亚单位疫苗,设计了ORFV B2L优势抗原表位截短重组蛋白,命名为HBBH并经原核表达和鉴定。以不同剂量HBBH不加或加不同佐剂,经滴鼻或颈部皮下注射免疫BALB/c小鼠,其中滴鼻共4组(HBBH 10μg、20μg、30μg和20μg+LTB),注射组(HBBH 20μg+201佐剂)。同时设ORFV组织灭活抗原+201佐剂注射免疫对照和空白对照,每组共免疫3次,每次间隔7 d。HBBH间接ELISA检测一免、二免、三免后7 d以及三免后14 d血清中ORFV IgG、IgA和三免后14 d肺灌洗液sIgA的抗体水平,取不同水平的IgG ELISA阳性血清检测细胞中和抗体效价。结果显示,表达并鉴定了具有良好反应原性的包含ORFV B2L蛋白优势抗原表位的截短重组蛋白HBBH。免疫小鼠试验结果表明,不同剂量HBBH通过滴鼻或颈部皮下注射均可刺激小鼠产生ORFV特异性IgG抗体,20μg HBBH+201佐剂注射免疫能最大刺激小鼠血清IgG抗体的产生。实验小鼠三免后14 d肺灌洗液特异性sIgA仅在HBBH滴鼻免疫剂量≥20μg的三个组中检测到,其中30μg HBBH组刺激小鼠产生了最高水平的sIgA。在同为20μg HBBH滴鼻免疫时,添加LTB佐剂刺激小鼠产生了更高水平的sIgA。表明一定剂量的HBBH+LTB佐剂滴鼻免疫可更好地刺激小鼠产生sIgA。HBBH间接ELISAIgG阳性血清的细胞中和抗体效价可高达1:128,且能中和4株ORFV分离株。结果表明,20μgHBBH+201佐剂注射免疫3次和20μg HBBH+LTB佐剂滴鼻免疫3次均能刺激BALB/c小鼠产生高水平的抗ORFV血清中和抗体IgG和黏膜抗体sIgA。本研究为ORFV B2L优势抗原表位截短重组蛋白作为亚单位疫苗提供了数据支持。

In order to research the sheep aphtha virus(ORFV)new subunit vaccine.Bioinformatics software were used to analyze the immune protective antigen epitopes of ORFV B2 L protein,for designing the truncated recombinant B2 L antigen epitope protein containing histidine label and flexible connecting area,the prokaryotic expression vector and recombinant ORFV truncated recombinant B2 L were constructed and expressed.BALB/c mice were immunized three times with different doses of HBBH(10 g,20 g,30 g)without or with different adjuvants(LTB or SEPEC 201 adjuvant)through nasal drop or neck subcutaneous injection.At the same time,ORFV tissue inactivated antigen containing SEPEC 201 adjuvant were set for control.ORFV Specific serum IgG,IgA and secretary IgA(sI gA)antibody levels were de-tected by indirect ELISA based on ORFV B2 L truncated recombinant protein HBBH.The truncated recombinant protein HBBH containing ORFV B2 L dominant antigen epitopes were efficiently expressed,which could bind to ORFV specific antibodies,indicating that the protein had good reactivity.The results showed that different doses of HBBH could stimulate the production of ORFV specific IgG antibody in mice by nasal drop or neck subcutaneous injection,and 20 g HBBH with SEPEC 201 adjuvant could stimulate the highest production of serum IgG antibody in mice.Lung lavage fluid specific sI gA was detected only in the three groups immuned with HBBH≥20 g of at 14 d post the third inmmune by nasal drop,among which the 30 g HBBH group stimulated the highest level of sI gA.When the HBBH nasal drop does were 20 g,the addition of LTB adjuvant stimulated mice to produce higher levels of sI gA.The results showed that a certain dose of HBBH plus LTB adjuvant by nasal immunization could better stimulate the production of sI gA in mice,however,the serum IgA produced by the HBBH immuned mice was maintained only for a short time.The highest titer of cell neutralizing antibody of the indrect ORFV HBBH ELISA positive IgG serum could reach 1∶128 and neutralize the 4 ORFV isolates used in this experiment.BALB/c mice were stimulated to produce high levels of anti-ORFV serum neutralizing IgG antibody by 20 g HBBH with SEPEC 201 adjuvant by neck subcutaneous injection immune and that of sI gA by 20 g HBBH with LTB adjuvant by nasal drop immune.This study provided data support for ORFV B2 L dominant antigen epitope truncated recombinant protein as subunit vaccine.