Porcine foot-and-mouth disease virus type O, type A fc polypeptide bivalent vaccine and its preparation method and application
A porcine foot-and-mouth disease virus and vaccine technology, applied in the field of medicine, can solve problems such as unsatisfactory requirements
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Embodiment 1
[0028] Embodiment 1, design and synthesis of porcine foot-and-mouth disease virus type A Fc polypeptide
[0029] 1. Design of porcine foot-and-mouth disease virus type A multi-epitope gene
[0030]According to the main antigen genes of three topological strains of porcine foot-and-mouth disease virus A (AF / 72, A / HB / WH / 09, A / GDMM / 2013), the 140-160 amino acid sequence and 200-213 amino acid sequence of the VP1 gene coding region were selected. The amino acid sequence is used as the epitope, and then the appropriate order is selected for tandem, that is, 140-160(A / GDMM / 2013)-GGSSGG-140-160(A / HBWH / 09)-GPLS-140-160(AF / 72)-GGGS-200-213 (A / GDMM / 2013), in order to prevent the formation of new epitopes after tandem epitopes, a spacer sequence is introduced between adjacent epitopes to ensure the independence of the epitopes. The spacers are GGSSGG, GPLS, and GGGS. The nucleotide sequence of the obtained porcine foot-and-mouth disease virus type A multi-epitope gene is shown in SEQ ...
Embodiment 2
[0035] Cloning and protein expression thereof of the gene of porcine foot-and-mouth disease virus O-type Fc polypeptide of embodiment 2, purification
[0036] 1. Optimization of porcine foot-and-mouth disease virus type O broad-spectrum multi-epitope fusion gene
[0037] Using bioinformatics code optimization software combined with multidisciplinary technologies such as immunology and biochemistry, the patent application with the publication number CN102675471A and the invention title "Porcine Foot-and-Mouth Disease Virus Type O Broad-spectrum Multi-epitope Recombinant Antigen and Its Application" was disclosed The nucleotide sequence encoding porcine foot-and-mouth disease virus type O broad-spectrum multi-epitope recombinant antigen was codon-optimized, and the optimized nucleotide sequence was shown as SEQ ID NO.4, named MEO-Fc. The sequence comparison analysis (Clustal W software and DNAMEN Version 9) of the genes before and after optimization showed that there were large ...
Embodiment 3
[0041] Embodiment 3, the preparation of porcine foot-and-mouth disease virus O type, A type Fc polypeptide bivalent vaccine
[0042] 1. Vaccine preparation
[0043] The two recombinant proteins MEA-Fc and MEO-Fc purified in Examples 1 and 2 were quantified by the Bio-Rad quantitative kit and then diluted to an appropriate concentration. The two proteins were prepared according to MEA-Fc:MEO-Fc=2: 1 (w / w) mixed, emulsified with oil adjuvant Montanide ISA206 (Seppic, France) according to 1:1 (w / w) into a vaccine preparation (W / O / W), 1ml per head, which contains porcine foot-and-mouth disease Viral O-type Fc polypeptide MEO-Fc 100 μg, porcine foot-and-mouth disease virus A-type Fc polypeptide MEA-Fc 200 μg.
[0044] 2. Immunity efficacy test:
[0045] The pigs used in the experiment weighed about 40kg, the O-type and A-type foot-and-mouth disease virus antibodies were less than 1:4 (results of liquid-phase blocking ELISA), and the 3ABC protein antibodies were negative (results ...
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