表达非洲猪瘟病毒P72蛋白复制缺陷型重组腺病毒的构建及鉴定

doi:10.11843/j.issn.0366-6964.2019.08.012

Abstract

Abstract: This study aimed to construct a defective recombinant adenovirus capable of expressing the P72 protein of African swine fever virus (ASFV). The ASFV B646L gene based on relevant China-SY18 isolate was synthesized. The synthetic B646L gene was cloned into transfer vector pENTR/D-TOPO. In order to construct a recombinant adenovirus, the recombination between the transfer vector pENTR/D-TOPO-ASFV-P72 and the backbone vector pAd/CMV/V5-DEST was done. The PacⅠ-linearized recombinant vector was transfected into 293A cell to produce recombinant adenovirus by serial passage. The results showed that the titers of the recombinant adenovirus Ad5-ASFV-P72 were 2.53×10⁹ IFU·mL^-1; the ASFV-P72 protein was expressed successfully in Vero cells by identification of the Indirect immunofluorescence assay and Western blot, and the expressed protein could reacted with ASFV standard positive serum specifically. Ad5-ASFV-P72 recombinant adenovirus was constructed successfully in the study. These findings not only lay the solid foundation for developing ASFV multi-gene recombinant adenoviral vector vaccine, but also provide a safe and effective antigen for the establishment serological diagnostic methods.

CLC Number:

S852.659.1

HU Yongxin, ZHAO Yonggang, ZHANG Yongqiang, LIU Fuxiao, FAN Xiaoxu, WU Xiaodong, WANG Zhiliang. Construction and Identification of Replication Defective Recombinant Adenovirus Expressing ASFV P72 Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1635-1641.

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References

[1] BLOME S, GABRIEL C, BEER M. Pathogenesis of African swine fever in domestic pigs and European wild boar[J].Virus Res, 2013, 173(1):122-130.
[2] 王志亮. 非洲猪瘟研究进展[J]. 中国动物检疫, 2011, 28(6):70-74.
WANG Z L.Advances in research of African swine fever[J]. Chinese Journal of Animal Health Inspection, 2011, 28(6):70-74. (in Chinese)
[3] 王清华, 任炜杰,包静月,等. 我国首例非洲猪瘟的确诊[J]. 中国动物检疫, 2018, 35(9):1-4.
WANG Q H, REN W J, BAO J Y, et al. The first outbreak of African swine fever was confirmed in China[J]. China Animal Health Inspection, 2018, 35(9):1-4. (in Chinese)
[4] LOPEZ-OTIN C, FREIJE J M P, PARRA E, et al. Mapping and sequence of the gene coding for protein p72, the major capsid protein of African swine fever virus[J]. Virology, 1990, 175(2):477-484.
[5] ZSAK L, ONISK D V, AFONSO C L, et al. Virulent African swine fever virus isolates are neutralized by swine immune serum and by monoclonal antibodies recognizing a 72-kDa viral protein[J].Virology, 1993, 196(2):596-602.
[6] GÓMEZ-PUERTAS P, RODRÍGUEZ F, OVIEDO J M, et al. Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization[J].J Virol, 1996, 70(8):5689-5694.
[7] GARCÍA-BARRENO B, SANZ A, NOGAL M L, et al. Monoclonal antibodies of African swine fever virus:antigenic differences among field virus isolates and viruses passaged in cell culture[J]. J Virol, 1986, 58(2):385-392.
[8] LEITAO A, MALUR A, COMELIS P, et al. Identification of a 25-amino acid sequence from the major African swine fever virus structural protein VP72 recognised by porcine cytotoxic T lymphocytes using a lipoprotein based expression system[J].J Virol Methods, 1998, 75(1):113-119.
[9] 戈胜强, 吴晓东,张志诚,等. 非洲猪瘟疫苗研究进展[J]. 畜牧兽医学报, 2016, 47(1):10-15.
GE S Q, WU X D, ZHANG Z C, et al. Progress in development of African swine fever vaccine[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(1):10-15. (in Chinese)
[10] TAKAMATSU H H, DENYER M S, LACASTA A, et al. Cellular immunity in ASFV responses[J]. Virus Res, 2013, 173(1):110-121.
[11] ALONSO F, DOMIÍNGUEZ J, VIÑUELA E, et al. African swine fever virus-specific cytotoxic T lymphocytes recognize the 32 kDa immediate early protein (vp32)[J]. Virus Res, 1997, 49(2):123-130.
[12] 龚振华,王丽萍,臧京帅,等. 非洲猪瘟病毒p54蛋白的高效表达及在ELISA中的应用[J]. 畜牧兽医学报, 2013, 44(11):1832-1837.
GONG Z H, WANG L P, ZANG J S, et al. Studies on the efficient expression of African swine fever virus p54 protein and its antigenic analysis in ELISA[J]. Acta Veterinaria et Zootechnica Sinica, 2013, 44(11):1832-1837. (in Chinese)
[13] MARTINS C L V, LAWMAN M J P, SCHOLL T, et al. African swine fever virus specific porcine cytotoxic T cell activity[J]. Arch Virol, 1993, 129(1-4):211-225.
[14] LOKHANDWALA S, WAGHELA S D, BRAY J, et al. Induction of robust immune responses in swineby using a cocktail of adenovirus-vectored African swine fever virus antigens[J]. Clin Vaccine Immunol, 2016, 23(11):888-900.
[15] MAEDA K, WEST K, HAYASAKA D, et al. Recombinant adenovirus vector vaccine induces stronger cytotoxic T-cell responses than recombinant vaccinia virus vector, plasmid DNA, or a combination of these[J].Viral Immunol, 2005, 18(4):657-667.
[16] TATSIS N, ERTL H C J. Adenoviruses as vaccine vectors[J]. Mol Ther, 2004, 10(4):616-629.
[17] KAMEN A, HENRY O. Development and optimization of an adenovirus production process[J].J Gene Med, 2004, 6(S1):S184-S192.
[18] GOGEV S, VANDERHEIJDEN M, LEMAIRE M, et al. Induction of protective immunity to bovine herpesvirus type 1 in cattle by intranasal administration of replication-defective human adenovirus type 5 expressing glycoprotein gC or gD[J]. Vaccine, 2002, 20(9-10):1451-1465.
[19] ZHU J G, HUANG X P, YANG Y P. Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways[J]. J Virol, 2007, 81(7):3170-3180.
[20] REVILLA Y, PÉREZ-NÚÑEZ D, RICHT J A. African swine fever virus biology and vaccine approaches[J]. Adv Virus Res, 2018, 100:41-74.
[21] LOKHANDWALA S, WAGHELA S D, BRAY J, et al. Adenovirus-vectored novel African swine fever virus antigens elicit robust immune responses in swine[J]. PLoS One, 2017, 12(5):e0177007.
[22] LOPERA-MADRID J, OSORIO J E, HE Y Q, et al. Safety and immunogenicity of mammalian cell derived and modified vaccinia Ankara vectored African swine fever subunit antigens in swine[J]. Vet Immunol Immunopathol, 2017, 185:20-33.

Construction and Identification of Replication Defective Recombinant Adenovirus Expressing ASFV P72 Protein

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