MDCK细胞悬浮培养驯化及其在增殖禽流感病毒H9亚型中的初步应用

doi:10.3969/j.issn.1674-7968.2020.01.019

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Abstract The pandemic of avian influenza poses a serious threat to the breeding industry and public health which could cause great losses in national economy. Currently, vaccination against avian influenza is the most effective preventive measure. There are many problems in the traditional chicken embryo technology, which is gradually replaced by cell technology. In order to evaluate the application of suspension culture technology in the production of inactivated cell-derived Avian influenza virus (AIV) vaccine, a well-defined adherent canine kidney cells (MDCK) was selected and acclimated to the suspension condition by gradually reducing serum. The AIV H9 subtype was inoculated into the adherent and suspended MDCK for adaptation culture. The cell-derived and chicken embryo-derived virus were prepared into inactivated vaccine. The specific pathogen free (SPF) chickens (Gallus gallus) were immunized and the immune effect of cell-derived and chicken embryo-derived AI inactivated vaccine was evaluated by serological method. The results showed that the MDCK cell density proliferated to 3.0×10⁶ cells/mL after 48 h of suspension culture, and reached to 5.0×10⁶ cells/mL after 72 h with good morphology, high viability and single suspension in culture medium. The data and cell status of shaking flask and 7.5 L bioreactor culture showed that the cell strain was suitable for suspension culture in a bioreactor. The AIV H9 subtype proliferated by suspension MDCK cells was used to prepare inactivated vaccine. After 21 d of immunization with SPF chickens, the geometric mean value of Hemagglutinatio inhibion (HI) in serum was more than 6(log₂), which was equivalent to the immune effect of chicken embryo-derived vaccine. The above experimental results suggest that the selected suspension culture MDCK cell line had the characteristics of rapid growth and stable proliferation, and could be used to proliferate AIV H9 subtype with high hemagglutination (HA) titer. The inactivated vaccine had good immunogenicity and could stimulate the body to produce antibodies with high titers. This study provides basic cytological data for large-scale production of AI and other viral vaccines.

Key words： Suspension culture technology MDCK cells Avian influenza virus Immunological evaluation

Received: 22 June 2019

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S381.3

Corresponding Authors: *cuiyan369@sina.com

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LIU Ping,CUI Yan. Conversion of MDCK Cells to Suspension Culture and Preliminary Application in the Proliferation of Avian influenza virus H9 Subtype[J]. 农业生物技术学报, 2020, 28(1): 184-190.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2020.01.019 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2020/V28/I1/184

[1] 吴培培, 唐应华, 褚轩, 等. 2016. 适应H9亚型禽流感病毒增殖的MDCK悬浮细胞株的驯化及其生物学特性[J]. 江苏农业学报. 32(6): 1377-1383.
(Wu P P, Tang Y H, Chu X, et al.2016. Domestication of MDCK suspension cell lines for the H9 subtype of Avain influenza virus proliferation and its biological characteristics evaluation[J]. Jiangsu Journal of Agricultural Sciences, 32(6): 1377-1383.)
[2] 中国兽药典委员会. 2015. 中华人民共和国兽药典•三部[M]. 北京: 中国农业出版社, pp. 3403-3404; 35-36.
(Chinese Pharmacopoeia Commission.2015. The People's Republic of China Volume Ⅲ[M]. China Agriculture Press, Beijing, China, pp. 3403-3404; 35-36.)
[3] Bock A, Sann H, Schulze-Horsel J, et al.2009. Growth behavior of number distributed adherent MDCK cells for optimization in microcarrier culture[J]. Biotechnology Progress, 25(6): 1717-1731
[4] Castro R, Fernandes P, Laske T, et al.2015. Production of Canine adenovirus type 2 in serum-free suspension cultures of MDCK cells[J]. Applied Microbiology and Biotechnology, 99(17): 7059-7068.
[5] Ernest M, Kamen A A.2015. Current and emerging cell culture manufacturing technologies for in?uenza vaccines[J]. Biomed Reserch Internation, 15: 11.
[6] Genzel Y, Behrendt I, Rödig J, et al.2013. CAP, a new human suspension cell line for In?uenza virus production[J]. Applied Microbiology and Biotechnology, 97(1): 111-122.
[7] Genzel Y, Olmer R M, Schäfer B, et al.2006. Wave microcarrier cultivation of MDCK cells for In?uenza virus production in serum containing and serum-free media[J]. Vaccine, 24(35-36): 6074-6087.
[8] Genzel Y, Vogel T, Buck J, et al.2014. High cell density cultivations by alternating tangential ?ow (ATF) perfusion for In?uenza A virus production using suspension cells[J.] Vaccine, 32(24): 2770-2781.
[9] Gregersen J P, Schmitt H J, Trusheim H, et al.2011. Safety of MDCK cellculture-based influenza vaccines[J]. Future Microbiology, 6(2): 143-152.
[10] Perdue M L, Arnold F, Li S, et al.2011. The future of cellculture-based in?uenza vaccine production[J]. Expert Review of Vaccines, 10(8): 1183-1194.
[11] Lohr V, Genzel Y, Behrendt I, et al.2010. A new MDCK suspension line cultivated in a fully deﬁned medium in stirred-tank and wave bioreactor[J]. Vaccine, 28(38): 6256-6264.
[12] Liu J, Shi X, Schwartz R, et al.2009. Use of MDCK cells for production of live attenuated influenza vaccine[J]. Vaccine, 27(46): 6460-6463.
[13] Mohler L, Flockerzi D, Sann H, et al.2005. Mathematical model of In?uenza A virus production in large-scale microcarrier culture[J]. Biotechnology and Bioengineering, 90(1): 46-58.
[14] Murakami S, Horimoto T, Mai le Q, et al.2008. Growth determinants for H5N1 influenza vaccine seed viruses in MDCK cells[J]. Journal of Virological Methods, 82(21): 10502-10509.
[15] Peschel B, Frentzel S, Laske T, et al.2013. Comparison of In?uenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line[J]. Vaccine, 31(48): 5693-5699.
[16] Tapia F, Vogel T, Genzel Y, et al.2014. Production of high-titer human In?uenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow ﬁber bioreactor[J]. Vaccine, 32(8): 1003-1011.
[17] Tree J A, Richardson C, Fooks A R, et al.2010. Comparison of large-scale mammalian cell culture systems with egg culture for the production of In?uenza virus A vaccine strains[J]. Biologicals, 38(5): 544-551.
[18] Wielink R, Kant-Eenbergen H C M, Harmsen M M, et al.2011. Adaptation of a Madin-Darby canine kidney cell line to suspension growth in serum-free media and comparison of its ability to produce Avian in?uenza virus to Vero and BHK21 cell lines[J]. Journal of Virological Methods, 171(1): 53-60.
[19] Youil R, Su Q, Toner T J, et al.2004. Comparative study of influenza virus replication in Vero and MDCK cell lines[J]. Journal of Virological Methods, 120(1): 23-31.