表达副鸡禽杆菌外膜蛋白的重组乳酸乳球菌的构建及其免疫原性研究
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摘要
为构建表达副鸡禽杆菌外膜蛋白的重组乳酸乳球菌,本试验按照乳酸乳球菌表达的偏好性,优化合成副鸡禽杆菌外膜蛋白p9的编码碱基序列,将其接入表达载体pNZ8149的多克隆位点并导入食品级乳酸乳球菌NZ3900。将重组乳酸乳球菌灭活后经肌肉注射免疫SPF鸡,免疫后攻毒测定乳酸乳球菌的保护效果,检测血清中Ig G抗体和鼻黏膜sIgA抗体水平并检验免疫效果。PCR结果显示,成功构建了含p9片段的重组乳酸乳球菌,Western-blot检测显示,p9片段可在重组乳酸乳球菌内表达。重组菌最佳诱导表达条件:Nisin浓度为20 ng/m L、诱导时间为4 h。鸡肌肉注射免疫后可产生p9蛋白特异性的血清Ig G和鼻黏膜sIgA抗体。乳酸乳球菌重组疫苗免疫鸡对副鸡禽杆菌攻毒的保护率为60%~80%。结果表明,本研究成功构建了重组乳酸乳球菌pNZ8149-p9/NZ3900,该重组菌作为免疫原免疫鸡,对副鸡禽杆菌攻毒具有较好的免疫保护作用。
To construct a recombinant Lactobacillus laccoccus that can express the outer membrane protein of Avibacterium paragallinarum(Apg).The p9 gene,which is part of encoding Apg outer membrane protein,was optimum synthesed according to the preference of Lactococcus lactis expression,and connected into the multiple cloning site of p NZ8149 expression vector.The recombinant vector was transformed into food-grade Lactobacillus lactobacillus NZ3900.Then,the recombinant Lactobacillus laccoccus was inactivated and injected SPF chicken by chest muscle.The test chickens were challenged by Apg to check the immune efficiency.Ig G and s Ig A were tested to check antibody level.The PCR result showed that the p9 gene was inserted into p NZ8149/NZ3900 successfully.Western-blot results showed the recombinant plasmid could be expressed in Lactobacillus lactobacillus,optimizing inducement time was4 h and the best concentration of inducer Nisin was 20 ng/m L.Serum Ig G and mucosa s Ig A could be detected after being inoculated by the vaccine made from L.lactis pNZ8149-p9/NZ3900.The injected chickens about from 60% to 80% were protected from Apg challenges.In conclusions,the recombinant Lactobacillus laccoccus with p NZ8149-p9/NZ3900 was successfully conducted,and the chickens inoculated with it showed perfect protection against Apg challenge.
To construct a recombinant Lactobacillus laccoccus that can express the outer membrane protein of Avibacterium paragallinarum(Apg).The p9 gene,which is part of encoding Apg outer membrane protein,was optimum synthesed according to the preference of Lactococcus lactis expression,and connected into the multiple cloning site of p NZ8149 expression vector.The recombinant vector was transformed into food-grade Lactobacillus lactobacillus NZ3900.Then,the recombinant Lactobacillus laccoccus was inactivated and injected SPF chicken by chest muscle.The test chickens were challenged by Apg to check the immune efficiency.Ig G and s Ig A were tested to check antibody level.The PCR result showed that the p9 gene was inserted into p NZ8149/NZ3900 successfully.Western-blot results showed the recombinant plasmid could be expressed in Lactobacillus lactobacillus,optimizing inducement time was4 h and the best concentration of inducer Nisin was 20 ng/m L.Serum Ig G and mucosa s Ig A could be detected after being inoculated by the vaccine made from L.lactis pNZ8149-p9/NZ3900.The injected chickens about from 60% to 80% were protected from Apg challenges.In conclusions,the recombinant Lactobacillus laccoccus with p NZ8149-p9/NZ3900 was successfully conducted,and the chickens inoculated with it showed perfect protection against Apg challenge.
引文
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[19] ZHOU J,WEI K,WANG C,et al.Oral immunization with Taishan Pinus massoniana pollen polysaccharide adjuvant with recombinant Lactococcus lactis expressing Proteus mirabilis ompA confers optimal protection in mice[J].Allergol Immunopathology,2017,45(5):496-505.
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[22]孙强正,熊衍文,叶长芸,等.食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达[J].微生物学报,2008,48(3):293-298.SUN Qiang-zheng,XIONG Yan-wen,YE Chang-yun,et al.Construction of a food-grade secretion expression vector and use it for reporter protein expression in Lactococcus lactis[J].Acta Microbiologica Sinica,2008,48(3):293-298.(in Chinese)
[2]路迎迎,路明华,陈小玲,等.B型副鸡禽杆菌安徽株的分离与鉴定[J].动物医学进展,2014,35(1):122-125.LU Ying-ying,LU Ming-hua,CHEN Xiao-ling,et al.Isolation and identification of an Avibacterium paragallinarum type B in Anhui Province[J].Progress in Veterinary Medicine,2014,35(1):122-125.(in Chinese)
[3]龚玉梅,张培君,孙惠玲,等.三型副鸡禽杆菌对SPF鸡的致病力试验[J].动物医学进展,2011,32(2):33-36.GONG Yu-mei,ZHANG Pei-jun,SUN Hui-ling,et al.Evaluation of three strains serotype of Avibacterium paragallinarum virulence to SPF chicken[J].Progress in Veterinary Medicine,2011,32(2):33-36.(in Chinese)
[4]龚玉梅,张培君,朱文革,等.鸡传染性鼻炎(三价)和新城疫二联灭活疫苗的研究[J].动物医学进展,2015,36(1):40-44.GONG Yu-mei,ZHANG Pei-jun,ZHU Wen-ge,et al.Studies on combined infectious coryza(serotype A+serotype B+serotype C)and Newcastle disease inactivated vaccine[J].Progress in Veterinary Medicine,2015,36(1):40-44.(in Chinese)
[5]凌云志,程小果,何福庆,等.鸡传染性鼻炎疫苗防控研究进展[J].动物医学进展,2016,37(8):94-98.LING Yun-zhi,CHENG Xiao-guo,HE Fu-qing,et al.Progress on prevention and control of infectious coryza by vaccination[J].Progress in Veterinary Medicine,2016,37(8):94-98.(in Chinese)
[6]彭小兵,蒋玉文,李旭妮,等.鸡传染性鼻炎灭活疫苗生产检验用副鸡禽杆菌Hpg-8株的保存期试验[J].中国兽药杂志,2013,47(6):14-16.PENG Xiao-bing,JIANG Yu-wen,LI Xu-ni,et al.Preservation period test of the Hpg-8 strain of A.paragallinarum master seed of infectious croyza inactivated vaccine[J].Chinese Journal of Veterinary Drug,2013,47(6):14-16.(in Chinese)
[7] JEONG J H,HA S C.Crystal structure of Nis I in a lipid free form,the Nisin immunity protein,from Lactococcus lactis[J].Antimicrobial Agents and Chemotherapy,2018,62(3):12-17.
[8] LIU L,ZHANG W,SONG Y,et al.Recombinant Lactococcus lactis co-expressing OmpH of an M cell-targeting ligand and IBDV-VP2 protein provide immunological protection in chickens[J].Vaccine,2018,36(5):729-735.
[9] ZHANG J S,GURI A,CORREDIG M,et al.Lactococcus lactis subsp.cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro[J].Food Research International,2016,90:147-153.
[10] SAEZ D,FERNANDEZ P,RIVERA A,et al.Oral immunization of mice with recombinant Lactococcus lactis expressing Cu,Zn superoxide dismutase of Brucella abortus triggers protective immunity[J].Vaccine,2012,30(7):1283-1290.
[11] HOU J,LU Y,LIU H Y,et al.Oral administration of Lactococcus lactis delivered heat shock protein65 attenuates atherosclerosis in low-density lipoprotein receptor-deficient mice[J].Vaccine,2011,29(24):4102-4109.
[12] ZHANG X J,DUAN G C,ZHANG R G,et al.Optimized expression of Helicobacter pylori ureB gene in the Lactococcus lactis Nisin-controlled gene expression(NICE)system and experimental study of its immunoreactivity[J].Current Microbiology,2009, 58(4):308-314.
[13] HOLO H,NES I F.High-frequency transformation by electroporation of Lactococcus lactis subsp.cremoris grown with glycine in osmotically stabilized media[J].Applied and Environmental Microbiology,1989, 55(12):3119-3123.
[14]赵成全,路明华,李淑芳,等.副鸡禽杆菌外膜蛋白GcbG的表达及免疫效果研究[J].中国兽医科学,2016,46(1):58-63.ZHAO Cheng-quan,LU Ming-hua,LI Shu-fang,et al.Expression and immune effect of GcbG from Avibacterium paragallinarum[J].Chinese Veterinary Science,2016,46(1):58-63.(in Chinese)
[15]王传文.表达空肠弯曲菌保护性抗原cfrA、cjaA基因的重组乳酸菌的构建及口服免疫研究[D].泰安:山东农业大学,2016.WANG Chuan-wen.Oral immunization of chickens with recombinant Lactococcus lactis expressing protective antigens cfrA and cjaA of Campylobacter jejuni[D].Taian:Shandong Agricultural University,2016.(in Chinese)
[16] PAUDEL S,HESS M,HESS C.Co-infection of Avibacterium paragallinarum and Gallibacterium anatis in specific-pathogen-free chickens complicates clinical signs of infectious coryza,which can be prevented by vaccination[J].Avian Diseases,2017,61(1):55-63.
[17]王宏俊.副鸡禽杆菌免疫优势模拟表位的鉴定及免疫原性分析[D].北京:中国农业大学,2007.WANG Hong-jun.Identification and immunogenicity of immunodominantmimotopes of Avibacterium paragallinarum[D].Beijing:China Agricultural University,2007.(in Chinese)
[18]王宏俊,龚玉梅,高亚萍,等.副鸡禽杆菌抗原表位的筛选与鉴定[J].中国兽医科学,2009,39(7):587-592.WANG Hong-jun,GONG Yu-mei,GAO Ya-ping,et al.Screening and identification of an epitope of Avibacterium paragallinarum from a phage display peptide library[J].Chinese Veterinary Science,2009,39(7):587-592.(in Chinese)
[19] ZHOU J,WEI K,WANG C,et al.Oral immunization with Taishan Pinus massoniana pollen polysaccharide adjuvant with recombinant Lactococcus lactis expressing Proteus mirabilis ompA confers optimal protection in mice[J].Allergol Immunopathology,2017,45(5):496-505.
[20] SINGH S K,ROEFFEN W,MISTARZ U H,et al.Construct design,production,and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine[J].Microbial Cell Factories,2017,16(1):97-108.
[21] CAO H P,WANG H N,YANG X,et al.Lactococcus lactis anchoring avian infectious bronchitis virus multi-epitope peptide EpiC induced specific immune responses in chickens[J].Bioscience,Biotechnology,and Biochemistry,2013,77(7):1499-1504.
[22]孙强正,熊衍文,叶长芸,等.食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达[J].微生物学报,2008,48(3):293-298.SUN Qiang-zheng,XIONG Yan-wen,YE Chang-yun,et al.Construction of a food-grade secretion expression vector and use it for reporter protein expression in Lactococcus lactis[J].Acta Microbiologica Sinica,2008,48(3):293-298.(in Chinese)