Preparation method, product and application of yolk antibody for resisting O-type and A-type foot-and-mouth disease viruses
An anti-foot-and-mouth disease virus and foot-and-mouth disease virus technology, applied in anti-virus immunoglobulin, egg-derived immunoglobulin, instruments, etc., can solve the problems of multi-antibody specificity and unsatisfactory stability.
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Embodiment 1
[0059] The preparation of embodiment 1 O type FMDV capsid protein (VP1)
[0060] 1. VP1 gene amplification
[0061] According to the O-type FMDV gene sequence published in the GenBank database, primers were designed to amplify the VP1 gene and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The primer sequences are shown in Table 1.
[0062] Table 1 Amplified VP1 gene primer sequence list
[0063]
[0064] Using O-type FMDV as a template, after PCR amplification and sequencing, the target fragment with the expected size was obtained, the size is 639bp, the PCR results are shown in figure 1 , and its nucleotide sequence is shown in SEQ ID NO.1 after sequencing.
[0065] 2. Construction and identification of recombinant plasmids
[0066] 2.1 Construction of recombinant plasmids
[0067] The DNA fragment was recovered, and the VP1 gene fragment was cloned according to the instructions of the pMD18-T vector to obtain the recombinant plasmid pMD18-T-VP1.
[0068] ...
Embodiment 2
[0082] The preparation of the whole antigen of embodiment 2 A type FMDV
[0083] 1. Cells, culture medium and virus species
[0084] The batch number of BHK-21 cells is BHK-21 / W / S-201201; the medium is low-serum medium MD611, purchased from Qingda Tianyi; MEM1611-030, processed by Yixing Saier Company; MEM, purchased from Hyclone Company. Newborn bovine serum was purchased from qualified suppliers, and was used after passing the internal control quality standard of newborn bovine serum of Zhongnongweite Biotechnology Co., Ltd. FMDV virus seed Re-A / WH / 09 / BF13SF2 suspension cell virus seed was provided by Zhongnong Weite Biotechnology Co., Ltd.
[0085] 2. BHK-21 cell preparation:
[0086] Resuscitate 1 cell, culture it statically in a T100 cell square flask, pass 2 to 3 generations, and when the cells grow to a dense monolayer, digest with trypsin, add an appropriate amount of cell culture medium, transfer to a 1000ml Erlenmeyer flask, and suspend in a 37°C incubator Culture...
Embodiment 3
[0093] The establishment of embodiment 3 optimal immunization strategies
[0094] 1. Selection of immune flocks
[0095] 100 7-week-old non-immune laying hens purchased from Lanzhou Zhengda Co., Ltd. were used for serum antibody detection with FMDV indirect ELISA detection kit, and a total of 80 chickens with antibody titers less than 1:8 were selected for subsequent experiments.
[0096] 2. Emulsification of different FMDV antigens
[0097] Respectively to the antigen solution (prepared in embodiment 2) containing 2ug / ml O type foot-and-mouth disease virus main capsid protein gene VP1 antigen (prepared in embodiment 1), containing 2ug / ml A type FMDV whole antigen and both according to volume ratio=1 : 1 In the mixed antigen solution obtained by mixing, add an equal weight of MONTANIDE ISA 206 vaccine adjuvant, 10000r / min, shear 3-5min in a shearing machine, emulsify into a homogeneous vaccine, and be used for immunizing animals.
[0098] 3. Determination of the immunization...
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