用口蹄疫A型病毒AF/72株的第3代乳鼠组织毒,通过乳鼠继续适应3代后,转适BHK-21细胞,获得该毒株的细胞毒AF/72/MF6/BF12,经测定其TCID50为10-8.0/mL;经RT-PCR获得其VPI基因序列,并与GenBank中的其他6株口蹄疫A型病毒株比对,同源性均大于85%;经无菌检验和外源病毒检验,纯净性达到兽用生物制品标准要求;经间接夹心ELISA测定,OD值均大于0.2,且该毒仅能被口蹄疫A型标准血清中和,具有型特异性;经紫外分光光度法测定其16S含量,均值为189 ng/mL,远大于22 ng/mL的国际标准.综合纯净性检验结果、特异性检验结果和16S含量,确定AF/72/MF6/BF12为口蹄疫A型病毒株AF/72的标准细胞毒.
杜进鑫, 王永录, 张永光, 方玉珍, 蒋守田, 吕建亮, 潘丽, 刘力宽
. 口蹄疫A型病毒AF/72株标准细胞毒株的研制[J]. 华北农学报, 2009
, 24(3)
: 74
-77
.
DOI: 10.7668/hbnxb.2009.03.016
Get the foot2and2mouth virus AF/ 72/ MF6/BF12by rat rejuvenation and BHK 221 cells adaption using FMD virus AF/ 72/ MF3the use of Karber method to measure AF/ 72/ MF6/BF12TCID50. its value was 8. 0;the main antigen VP1 gene sequence was got by RT2PCR with 6 type A strains of FMDV in GenBank,homology was higher than 85 %.By the sterile examination,other exogenous virus test,its purityof AF/ 72/ MF6/BF12conform to Biological Products Standards for Animal Use;Indirect Sandwich ELISA OD Values greater than 0. 2,This virus was only neutralized by the reference serum of FMD type A in line with the specific;146S antigen content was measured by ultraviolet spectrophotometer,The average value was much higher than 22 ng/ mL of international standards. AF/ 72/ MF6/BF12was identified as reference cellular strain of AF/ 72 of FMD type A virus integrating many parameters.
[1] 王在时.国内外标准物质研制现状及发展趋势[C]//国家兽用标准物质研讨会会议资料,2007:1-12.
[2] 殷震,刘景华.动物病毒学[M].北京:科学出版社,1997.
[3] Cartwright B,Chapman W G,Brown F.Serological and im-manological relationships between the 146S and 12S particles of Foot-and-mouth disease virus[J].J Gen Virol,1980,50:369-375.
[5] Cowan K M.Antibody response to viral antigens[J].Adv Im-munol,1973,17:195-255.
[6] 李忠润,刘湘涛,胡弘博,等.口蹄疫病毒12S亚单位抗原免疫学性质的研究[C]//中国畜牧兽医学会口蹄疫学分会第七次全国口蹄疫学术研讨会论文集.兰州:中国农业科学院兰州兽医研究所,1997:186-189.
[7] Crowther J R,Reckziegel P O,Prado J A.Quantification of whole virus particles(146S)of Foot-and-mouth disease Virus in the presence of virus subunits(12S),using monoclonal an-tibodies in a sandwich ELISA[J].Vaccine,1995,13:1064-1075.
[8] Doel T R,Chong W K T.Comparative immunogenicity of 146S,75S and 12S particles of Foot-and-mouth disease Virus[J].Archives of Virology,1982,73:185-191.
[9] Black L,Nicholls M J,Rweyemamu M M,et al.Foot-and-nlouth disease vaccination:a multifactofial study of the influ-ence of antigen dose and potentially competitive immunogens on the response of cattle of different ages[J].Res Vet Sci,1986,40(3):303-7.
[10] Pay T W,Hingley P J.Foot and mouth disease vaccine po-tency tests in cattle:the interrelationship of antigen dose,serum neutralizing antibody response and protection from challenge[J].Vaccine,1993,11(3):392.
[11] 肖昭彭,杨福荣.用紫外分光光度计检测口蹄疫病毒的研究[J].中国兽医科技,1997,27(7):23-24.
[12] Bachrach H L.The determination of the sedimentation con-stant of a homogeneous component having the characteristics of the foot-and-mouth disease virus[J].Amer J Vet Res,1952,13:13-16.
[13] Duchesne M,Guerche J,Leqrand B,et al.The use of highly concentrated purified(by a large scale method)and long term liquid nitrogen stored foot-and-mouth disease viruses for the preparation of vaccines:physico-chemical quality controls and potency tests after storage[J].Dev Biol Stand,1981,50:249-259.
[14] Doel T R,Collen T.Qualitative assessment of 146S particles of foot-and-mouth disease virus in preparations destined for vaccines[J].J Biol Standard,1982,10:69-81.
[15] Reed L J,Muench H.A simple method of estimating fifty percentend points[J].Am J Hyg,1938,27:493-497.
[16] Pay T W F,Hingley P L.Correlation of 140S antigen dose with the serum neutralizing antibody response and the level of protection induced in cattle by foot-and-mouth disease vaccines[J].Vaccine,1987,5:60-64.
