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1 生物工程学报 Chin J Biotech 2009, July 25; 25(7): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 动物及兽医生物技术 C3d-M28 gc 樊惠英 1*, 刘中勇 2*, 佟铁铸 3,4, 刘星 3, 郭爱珍 1, , , , : 研究补体 C3d 的受体结合功能区 (M28) 对伪狂犬病毒 gc 基因 DNA 疫苗免疫增强作用将 4 拷贝的 M28 基因与伪狂犬病毒 gc 基因串联后, 克隆到载体 pcdna3.1 中, 构建融合表达的重组质粒 (sgc-m28 4 ) BALB/c 小鼠免疫试验表明, sgc-m28 4 免疫组比单独表达伪狂犬病毒 gc 蛋白的重组质粒 (sgc) 免疫组产生的 ELISA 抗体高 17 倍, 对致死剂量 (316 LD 50 ) 伪狂犬病毒攻毒的保护率提高了 63% gc 基因与 M28 基因融合表达诱导产生的 IL-4 水平接近了伪狂犬灭活疫苗免疫组的产生的 IL-4 水平, 显著增强了基于 Th2 途径的体液免疫反应 : 补体 C3d, gc 糖蛋白, 伪狂犬病毒, C3d 受体结合功能区 (M28), 分子免疫佐剂 4 C3d-M28 enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus Huiying Fan 1*, Zhongyong Liu 2*, Tiezhu Tong 3,4, Xing Liu 3, and Aizhen Guo 4 1 College of Veterinary Medicine, South China Agricultural University, Guangzhou , China 2 Guangdong Entre-exit Inspection and Quarantine Bureau, Guangzhou , China 3 Huizhou Entre-exit Inspection and Quarantine Bureau, Huizhou , China 4 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan , China Abstract: We studied the immunogenicity of pseudorabies virus gc DNA vaccination by fusing the murine complement C3d receptor binding domain. First, pseudorabies virus gc gene was linked to four copies of C3d receptor binding domain (M28 4 ), and then cloned into the vector pcdna3.1 to construct the recombinant plasmid sgc-m284. Through the experiment of immunized BALB/c mice, we found that the enzyme linked immunosorbent assay (ELISA) antibody titer for sgc-m284 was 17-fold higher than that for sgc alone, and protective rate of mice was augmented from 25% to 88% after lethal dose PrV (316 LD 50 ) challenge. In addition, the IL-4 levels for sgc-m28 4 immunization approached that for the pseudorabies virus inactivated vaccine. In conclusion, we demonstrated murine C3d receptor binding domain fusion significantly increased Th2-biased immune response by inducing IL-4 production. Keywords: C3d, gc protein, pseudorabies virus, complement C3d receptor binding domain M28, molecular adjuvants Received: January 19, 2009; Accepted: May 22, 2009 Supported by: National Natural Sciences Foundation of China (No ). Corresponding author: Aizhen Guo. Tel: ; Fax: ; aizhen@mail.hzau.edu.cn * These authors contributed equally to this study. 国家自然科学基金项目 (No ) 资助

2 988 ISSN CN /Q Chin J Biotech July 25, 2009 Vol.25 No.7 (Pseudorabies), [1,2] gb gc gd, gb gc gd, [3], DNA [4], GM-CSF [5] CpG [6] IL-2 [7] IFN-γ [8], C3d [9], C3d [10] C3d B 2 CD21 CD21 B CD19, CD21, CD19 [11] B, B (BCR), C3d CD19 [12], B CD21, 2(CR2) C3d (M28) C [13] C3d CR2 M28 gc DNA 1.2 方法 M28 M28 C3 (GenBank Accession No. NM_009778), C3d puc-19 ( ) PCR, 1 Toss [11], puc-19-linke 4 M28 puc19-4(m28) gc gc psfvc1.5 ( ), GenBank gc (GenBank Accession No. AF158090) gc ( ), Bgl II BamH I, 1 PCR Bgl II BamH I BamH I pcdna3.1-tpa, sgc sgc-m28 4 Bgl II BamH I puc19-4(m28), BamH I sgc M28 gc, sgc- M28 4 ( 1) LipofectAMINE 2000 (Invitrogen, USA) PK-15, G418, 30 μl SDS-PAGE, gc Western blotting 1 材料与方法 1.1 细胞毒株和质粒 (PrV) A(Ea) DH5α PK-15 linker(g4s2) puc-19-linker Bello tpa (Tissue plasminogen activator- ) pcdna3.1-tpa 图 1 重组表达质粒的结构示意图 Fig. 1 Schematic diagram of the plasmids expressing sgc-m28 4 and sgc.

: C3d-M28 gc 989 表 1 用于 M28 和 gc 基因扩增的引物序列 Table 1 Primer pairs used to amplify the M28 and gc gene Primer name M28 (sense primer) M28 (antisense primer gc (sense primer) gc (antisense primer Primer

3 : C3d-M28 gc 989 表 1 用于 M28 和 gc 基因扩增的引物序列 Table 1 Primer pairs used to amplify the M28 and gc gene Primer name M28 (sense primer) M28 (antisense primer gc (sense primer) gc (antisense primer Primer sequence (5 3 ) ACGCGTCGACCATGGATCCCAACA TCGGGATCCGGCCTAGGATGTG TTTAGATCTCCGTCGTCCACGACGGCGCTCGGC TTTGGATCCGGGCAGCTGCGAGGGCATGCCCTC ~8 BALB/c 4, 10, pcdna3.1 sgc sgc- M28 4 3, 3, 100 μl 100 μg PBS 2, 4, 2, 100 μl , ELISA 8, 100 μl TCID 50 ( 316 LD 50 ),, ELISA ELISA, ELISA 100, 100 μl, : 37 o C 1 h, HRP ( )100 μl/, 37 o C 1 h, TMB 100 μl, 15 min, 50 μl, 630 nm OD PBS, PBS OD IFN-γ IL-4 8, ( / ), 20 μl PrV( TCID 50, 0.1 ml) 72 h, IFN-γ IL-4 (Biosourse, USA) IFN-γ IL t P < 0.05, P < 结果 2.1 重组质粒的表达 gc(sgc) M28 gc (sgc-m28 4 ), PK-15,, 30 μl 上 SDS-PAGE Western blotting sgc sgc-m kd 60 kd ( 2) sgc sgc-m28 4, 4 M28 图 2 Western blotting 检测 pcdna3.1+ sgc 和 sgc-m28 4 在 PK-15 细胞中的表达 Fig. 2 Expression of pcdna3.1+, sgc and sgc-m28 4 in transfected PK-15 cells was detected by Western blotting with the antibody to gc protein. 1: blank vector pcdna3.1+; 2: sgc-m28 4 encoding about 60 kd fusion protein of sgc-m28 4 ; 3: sgc encoding about 44 kd ectodomain of gc protein. 2.2 PrV gc 蛋白 ELISA 抗体的检测 ELISA ELISA

990 ISSN1000-3061 CN11-1998/Q Chin J Biotech July 25, 2009 Vol.25 No.7, 2, sgc-m28 4 sgc (P < 0.01), sgc 1:100, sgc sgc-m28 4 8 1:600 1: 9549, 1:39 810( 3) 图 3 首免后不同免疫组诱导的抗 gc IgG ELISA 抗体检测 Fig.

3 细胞因子 IL-4 和 IFN-γ 检测, IL-4 IFN-γ ELISA IL-4 IFN-γ, IL-4 IFN-γ IFN-γ Th1, IL-4 Th2 2 ( 4): IL-4 (2000 pg/ml), sgc IL-4 (574 pg/ml) M28 IL-4, IL-4 (1550 pg/ml) sgc (574 pg/ml) (P<0.

4 990 ISSN CN /Q Chin J Biotech July 25, 2009 Vol.25 No.7, 2, sgc-m28 4 sgc (P < 0.01), sgc 1:100, sgc sgc-m :600 1: 9549, 1:39 810( 3) 图 3 首免后不同免疫组诱导的抗 gc IgG ELISA 抗体检测 Fig. 3 Anti-gC IgG growth in mice after intramuscularly inoculation of various plasmids or inactivated vaccine. 2.3 细胞因子 IL-4 和 IFN-γ 检测, IL-4 IFN-γ ELISA IL-4 IFN-γ, IL-4 IFN-γ IFN-γ Th1, IL-4 Th2 2 ( 4): IL-4 (2000 pg/ml), sgc IL-4 (574 pg/ml) M28 IL-4, IL-4 (1550 pg/ml) sgc (574 pg/ml) (P<0.05) sgc-mm284 IL-4 IL-4( 4A) sgc sgc-mm284 IFN-γ (697 pg/ml), IFN-γ (239 pg/ml) (P < 0.01) ( 4B) 2.4 攻毒保护试验 3 2, μl 10 5 TCID 50 ( 316 LD 50 ) PrV Ea 图 4 免疫组脾细胞 IL-4 IFN-γ 水平检测 Fig. 4 Levels of IL-4 (A) and IFN-γ (B) from splenocytes in mice immunized with gc encoding plasmids or inactivated vaccine. 表 2 攻毒后小鼠的存活情况 Table 2 Survival of the vaccinated mice after challenged with lethal PrV Ea strain a Group Survival (No. of mice) Protection rate (%) Clinical sign b sgc 2/ sgc-mm284 7/8 88 +/ Inactivated vaccine 8/8 100 Vector 0/8 0 + a: the immunized mice were challenged with 316 LD 50 of PrV Ea strain; b: mice showing visible signs of infection were recorded as +; mice not showing visible signs of infection were recorded as ; one to three among eight mice showing visible signs of infection were recorded as +/., sgc,,,, 3 4

5 : C3d-M28 gc 991 sgc-mm ,, 88% sgc 4 2, 6~8 4, 25%( 2) 3 讨论, C3d [10 12] C3d, C3d CR2 M28 C3d CR2 M28, sgc-mm284 ELISA (1: 9549) sgc (1: 600) 17 ( 3) sgc-m284 (SI) sgc, sgc sgc-mm284 IFN-γ (697 pg/ml), IFN-γ (239 pg/ml), sgc gc-mm284, 1: IL-4 (2000 pg/ml) sgc-mm284 ELISA (1: 9549) sgc 6, 2, 25%, sgc-mm284 1, M28 gc Th2, sgc-mm284 Th1/Th2, C3d CR2 M28 gc, gc B BCR, M28 B CR2 CD21 [13] BCR CD21/CD19 BCR sgc-mm284 IL-4 (1550 pg/ml) sgc IL-4 (574 pg/ml),, C3d CR2 M28 gc gc, REFERENCES [1] Bouma A. Determination of the effectiveness of pseudorabies marker vaccines in experiments and field trials. Biologicals, 2005, 33: [2] Dory D, Torche AM, Beven V, et al. Effective protection of pigs against lethal pseudorabies virus infection after a single injection of low-dose Sindbis-derived plasmids encoding PrV gb, gc and gd glycoproteins. Vaccine, 2005, 23: [3] Van Rooij EM, Moonen-Leusen HW, de Visser YE, et al. A DNA vaccine coding for gb and gd of pseudorabies virus (suid herpes type 1) primes the immune system in the presence of maternal immunity more efficiently than conventional vaccines. Vaccine, 2006, 24: [4] Dufour V, De Boisseson C. Use of a Sindbis virus DNA-based expression vector for induction of protective immunity against pseudorabies virus in pigs. Vet Immuno Immunopathol, 2003, 93: [5] Yoon HA, Aleyas AG, George JA, et al. Cytokine GM-CSF genetic adjuvant facilitates prophylactic DNA vaccine against pseudorabiesvirus through enhanced immune responses. Microbiol Immunol, 2006, 50: [6] Dory D, Beven V, Torche AM, et al. CpG motif in ATCGAT hexamer improves DNA-vaccine efficiency against lethal pseudorabies virus infection in pigs. Vaccine, 2005, 23: [7] Bu J, Song Y, Rompato G, et al. Co-delivery of IL-2 or liposomes argument the responses of mice to a DNA vaccine for pseudorabies virus IE180. Comp Immunol Microbiol Infect Dis, 2003, 26: [8] Tudor D, Riffault S, Carrat C, et al. Type I IFN modulates the immune response induced by DNA vaccination to pseudorabies virus glycoprotein C. Virology, 2001, 286: [9] Fischer T, Buttner M, Rziha HJ. T helper 1-type cytokine transcription in peripheral blood mononuclear cells of pseudorabies virus (Suid herpesvirus 1)-primed swine indicates efficient immunization. Immunology, 2000, 101: [10] Wang L, Sunyer JO, Bello LJ. Immunogenicity of a bovine viral diarrhea virus E2-C3d fusion protein containing a bovine homolog of C3d. Dev Comp Immunol,

6 992 ISSN CN /Q Chin J Biotech July 25, 2009 Vol.25 No , 29: [11] Mitchell JA, Green TD, Bright RA, et al. Induction of heterosubtypic immunity to influenza A virus using a DNA vaccine expressing hemagglutinin-c3d fusion proteins. Vaccine, 2003, 21: [12] Green TD, Montefiori DC, Ross TM. Enhancement of antibodies to the human immunodeficiency virus type 1 envelope by using the molecular adjuvant C3d. J Virol, 2003, 77: [13] Wang L, Sunyer JO, Bello LJ. Fusion to C3d enhances the immunogenicity of the E2 glycoprotein of type 2 bovine viral diarrhea virus. J Virol, 2004, 78: 生物工程学报将结合第七届中国酶工程学术研讨会出版酶工程专刊 ~ ~ , ( cjbcn/ch/index.aspx)/ / 2., / / 3. ( ) 4. ligaox2003@yahoo.com.cn cjb@im.ac.cn 1 3 B

Microsoft Word - 052008-743-090065.doc

Microsoft Word - 052008-743-090065.doc 中国科学 C 辑 : 生命科学 2009 年第 39 卷第 6 期 : 534 ~ 541 www.scichina.com life.scichina.com SCIENCE IN CHINA PRESS 人 - 禽双价流感新型 DNA 疫苗构建及免疫保护实验研究南文龙 12, 金宁一 2*, 鲁会军 2, 赵翠青 12, 田