嗜水气单胞菌外膜蛋白(OMP)在乳酸乳球菌中的表达及其对BALB/c小鼠的免疫保护效果

乳酸球菌(Lactococcus lactis)是一种安全级微生物,为活载体疫苗传递抗原的理想选择.嗜水气单胞菌(A eromonas hydrophila,Ah)能引起鱼类等多种动物的败血病,因其血清型众多,寻找共同保护性抗原是进行疫苗研究的重点.为探究嗜水气单胞菌AS1.927株外膜蛋白(OMP)在乳酸乳球菌中的表达和检测其表达产物对小鼠的免疫保护效果,本研究将己克隆的ompA基因经BamH Ⅰ和Xba Ⅰ双酶切后连接载体pNZ8048,转化乳酸乳球菌NZ9000,nisin诱导表达,并进行SDS-PAGE分析鉴定.结果显示,重组基因工程菌L.lactispNZ-ompA]表达的融合蛋白分子量约为36.2 kD,成熟蛋白分子量约为33.7 kD.将重组基因工程菌L.lac tis pNZ-ompA]口服免疫BALB/c小鼠(Mus musculus),用放射免疫法检测末次免疫一周后的各组小鼠肠粘膜分泌型免疫球蛋白A(sIgA)的水平以及用间接ELISA法检测小鼠血清特异性抗体IgG,结果发现,该重组基因工程菌能显著提高sIgA的分泌(P＜0.05)；同时在小鼠血清中的特异性抗体含量也显著高于对照组(P＜0.05).末次免疫两周后用100 LD50的Ah AS 1.927(3.3×105 cfu/mL)腹腔注射攻击小鼠,口服重组菌对小鼠的相对免疫保护力(relative percent survival,RPS)为87.5％,表明重组基因工程菌可诱导小鼠对AhAS1.927产生一定的免疫保护作用.该结果将为开发安全有效的口服基因工程鱼用疫苗提供一定的实验基础.

The Expression of Outer Membrane Protein (OMP) from A eromonas hydrophila in Lactococcus lactis and the Immunoprotection in BALB/c Mice

As a safety microorganism, Lactococcus lactis is an ideal live vector vaccine candidate for carrying antigen. Aeromonas hydrophila cause hemorrhagic septicemia in many kinds of animals including fish. It is important to find the common protective antigens in vaccine research work, because of the numerous serotype. To explore how the outer membrane protein (OMP) from Aeromonas hydrophila (Ah) AS1.927 strain expressed in the L. lactis and its immunological protection of the expressed OMP, a cloned ompA gene was inserted into pNZ8048 vector and expressed in the L. lactis NZ9000 using nisin induction in this study . The expressed OMP was estimated by migration in 10% sodium dodecyl sulphate-polyarylamide gel electrophoresis (SDS-PAGE). Results showed that the size of the fusion protein was about 36.2 kD, and the mature protein was about 33.7 kD. BALB/c mice (Mus musculus) were orally inoculated with the engineering bacteria L.lactis pNZ-ompA]. Intestinal secretory immunoglobulin A (sIgA) was determined by double antibody radioimmunoassay and serum IgG was detected by enzymelinked immunosorbent assay (ELISA) at one week post-vaccination,the results indicated that mice vaccinated with L.lactis pNZ-ompA] significantly increased sIgA level and antigen-specific IgG level in the serum (P<0.05) than that of untreated control group . Besides, on the 14th day after the last immunization, the mice were challenged with 3.3×105 cfu/mL of live Ah AS1.927 (100 LD50), showing that the immunized group had 87.5% relative percent survival (RPS). This indicated that oral immunization of L.lactis pNZ-ompA] can protectively initiate an immune response upon. It provides a technical basis for developing efficient oral gene engineering vaccine against fish Ah.