重组PCV3 Cap蛋白植物乳杆菌的构建与表达验证
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摘要
构建3型猪圆环病毒(PCV3)Cap蛋白的重组植物乳杆菌并进行验证。通过PCR方法扩增含1320信号肽和DCpep优化的PCV3 Cap基因(PCV3D),使用无缝克隆技术连接到pSIP411载体中,电转入克隆宿主乳球菌NZ3900中,提取测序结果正确的重组表达质粒NZ3900:pSIP411-PCV3D再次电转入植物乳杆菌Lp12,制备重组菌并验证其表达。结果显示:成功扩增出目的条带为789 bp的经过修饰和优化的PCV3 Cap基因(PCV3D)片段,重组表达质粒电转入植物乳杆菌Lp12,成功构建重组植物乳杆菌Lp12:pSIP411-PCV3D。Western blot、流式细胞术等方法检测重组蛋白表达,获得阳性结果,阳性率为47.9%;免疫荧光进一步表明重组蛋白能够在细胞中表达。本试验成功构建了1株表达PCV3 Cap蛋白的植物乳杆菌,为PCV3口服候选疫苗的开发研究提供依据。
Recombinant Lactobacillus plantarum with type 3 porcine circovirus(PCV3) Cap protein was constructed and validated.The 1320 signal peptide and DCpep-optimized PCV3 Cap gene(PCV3 D) were amplified by PCR,ligated into the pSIP411 vector using the seamless cloning technique,and electroporated into the cloning host Lactococcus NZ3900 to extract the recombinant expression plasmid NZ3900:pSIP411-PCV3 D.It was again electroporated into Lactobacillus plantarum Lp12,and recombinant Lactobacillus plantarum were prepared and their expression was verified.A modified and optimized PCV3 Cap gene(PCV3 D) fragment with a target band of 789 bp was successfully amplified.The recombinant expression plasmid was electroporated into Lactobacillus plantarum Lp12,and recombinant Lactobacillus plantarum Lp12:pSIP411-PCV3 D was successfully constructed.Western blot,flow cytometry and other methods were used to detect the expression of recombinant protein,and the positive result was obtained.The positive rate was 47.9%.Immunofluorescence further indicated that the recombinant protein is capable of being expressed in cells.In this study,a strain of Lactobacillus plantarum expressing PCV3 Cap protein was successfully constructed,which provided a theoretical basis for the development of PCV3 oral vaccine candidate.
Recombinant Lactobacillus plantarum with type 3 porcine circovirus(PCV3) Cap protein was constructed and validated.The 1320 signal peptide and DCpep-optimized PCV3 Cap gene(PCV3 D) were amplified by PCR,ligated into the pSIP411 vector using the seamless cloning technique,and electroporated into the cloning host Lactococcus NZ3900 to extract the recombinant expression plasmid NZ3900:pSIP411-PCV3 D.It was again electroporated into Lactobacillus plantarum Lp12,and recombinant Lactobacillus plantarum were prepared and their expression was verified.A modified and optimized PCV3 Cap gene(PCV3 D) fragment with a target band of 789 bp was successfully amplified.The recombinant expression plasmid was electroporated into Lactobacillus plantarum Lp12,and recombinant Lactobacillus plantarum Lp12:pSIP411-PCV3 D was successfully constructed.Western blot,flow cytometry and other methods were used to detect the expression of recombinant protein,and the positive result was obtained.The positive rate was 47.9%.Immunofluorescence further indicated that the recombinant protein is capable of being expressed in cells.In this study,a strain of Lactobacillus plantarum expressing PCV3 Cap protein was successfully constructed,which provided a theoretical basis for the development of PCV3 oral vaccine candidate.
引文
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[2] ALLAN G M,MCNEILLY E,KENNEDY S,et al.PCV-2-associated PDNS in Northern Ireland in 1990:Porcine dermatitis and nephropathy syndrome[J].Vet Rec,2000,146(24):711-712.
[3] PHAN T G,GIANNITTI F,ROSSOW S,et al.Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virol J,2016,13(1):184.
[4] SARAIVA G L,VIDIGAL P M P,FIETTO J L R,et al.Evolutionary analysis of porcine circovirus 3 (PCV3) indicates an ancient origin for its current strains and a worldwide dispersion[J].Virus Genes,2018,54(3):376-384.
[5] 郭奎,徐朋丽,赵宇,等.PCV3河南株Cap基因的克隆、测序及其在大肠杆菌中的表达[J].中国兽医学报,2018,38(11):2063-2067.
[6] KU X,CHEN F,LI P,et al.Identification and genetic characterization of porcine circovirus type 3 in China[J].Transbound Emerg Dis,2017,64(3):703-708.
[7] JIANG H,WANG D,WANG J,et al.Induction of porcine dermatitis and nephropathy syndrome in piglets by infection with porcine circovirus type 3[J].J Virol,2019,93(4):pii:e02045-18.
[8] FREDRIKSEN L,MATHIESEN G,SIOUD M,et al.Cell wall anchoring of the 37-kilodalton oncofetal antigen by Lactobacillus plantarum for mucosal cancer vaccine delivery[J].Appl Environ Microbiol,2010,76(21):7359-7362.
[9] LANDETE J M.A review of food-grade vectors in lactic acid bacteria:from the laboratory to their application[J].Crit Rev Biotechnol,2017,37(3):296-308.
[10] HOU X L,YU L Y,LIU J,et al.Surface-displayed porcine epidemic diarrhea viral (PEDV) antigens on lactic acid bacteria[J].Vaccine,2007,26(1):24-31.
[11] YANG W T,YANG G L,SHI S H,et al.Protection of chickens against H9N2 avian influenza virus challenge with recombinant Lactobacillus plantarum expressing conserved antigens[J].Appl Microbiol Biotechnol,2017,101(11):4593-4603.
[12] 倪能能,王新珂,赖强,等.猪圆环病毒2型ORF2基因重组植物乳杆菌的构建及其免疫原性分析[J].中国畜牧兽医,2017,44(8):2408-2416.
[13] PALINSKI R,PI?EYRO P,SHANG P,et al.A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].J Virol,2017,91(1):pii:e01879-16.
[14] PETROVA M I,VAN DEN BROEK M F L,SPACOVA I,et al.Engineering Lactobacillus rhamnosus GG and GR-1 to express HIV-inhibiting griffithsin[J].Int J Antimicrob Agents,2018,52(5):599-607.
[15] HUANG K Y,YANG G L,JIN Y B,et al.Construction and immunogenicity analysis of Lactobacillus plantarum expressing a porcine epidemic diarrhea virus S gene fused to a DC-targeting peptide[J].Virus Res,2018,247:84-93.
[16] MARCOBAL A,LIU X,ZHANG W,et al.Expression of human immunodeficiency virus type 1 neutralizing antibody fragments using human vaginal Lactobacillus[J].AIDS Res Hum Retroviruses,2016,32(10/11):964-971.
[17] SHI S H,YANG W T,YANG G L,et al.Lactobacillus plantarum vaccine vector expressing hemagglutinin provides protection against H9N2 challenge infection[J].Virus Res,2016,211:46-57.
[18] QIAO X,LI G,WANG X,et al.Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice[J].BMC Microbiol,2009(9):249.
[19] KARLSKAS I L,MAUDAL K,AXELSSON L,et al.Heterologous protein secretion in Lactobacilli with modified pSIP vectors[J].PLoS One,2014,9(3):e91125.
[20] NGUYEN T T,NGUYEN H M,GEIGER B,et al.Heterologous expression of a recombinant lactobacillal beta-galactosidase in Lactobacillus plantarum:effect of different parameters on the sakacin P-based expression system[J].Microb Cell Fact,2015,14:30.
[21] SORVIG E,MATHIESEN G,NATERSTAD K,et al.High-level,inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors[J].Microbiology,2005,151(Pt 7):2439-2449.
[22] CORTES-PEREZ N G,LEFèVRE F,CORTHIER G,et al.Influence of the route of immunization and the nature of the bacterial vector on immunogenicity of mucosal vaccines based on lactic acid bacteria[J].Vaccine,2007,25(36):6581-6588.