Method of diagnosis of foot and mouth disease and the diagnostic kit
a technology of foot and mouth disease and kit, which is applied in the direction of instruments, biochemistry apparatus and processes, material analysis, etc., can solve the problems of loss of milk, high financial loss as a result of fmd, and extremely contagious disease, and achieve the effect of diagnosing infection
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example 1
[0072] Preparation of Recombinant FMDV VP1 Antigen
[0073] A. Construction of FMDV VP1 Expression Vectors
[0074] (i) Construction of Synthetic VP1 Gene
[0075] VP1 protein of Foot and Mouth Disease virus Taiwan Type O 97 sequence was retrieved from NCBI GenBank data and oligonucleotides for syntheictc gene were synthesized at ResGen (Huntsville, Ala.). In the synthetic oligonucleotides, the native FMDV codons were altered to conform to E. coli codon bias in an effort to increase expression levels of the recombinant protein in E. coli. See, for example, M. Gouy and C. Gautier, Nucleic Acids Research 10:7055 (1982); H. Grosjean and W. Fiers, Gene 18:199 (1982); J. Watson et al. (eds.), Molecular Biology of the Gene, 4th Ed., Benjamin Kumming Publishing Co., pp. 440 (1987). The recursive PCR method was used to assemble the oligonucleotides into full VP1 gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When anneal...
example 2
[0093] Preparation of Recombinant FMDV 2C Antigen
[0094] A. Construction of FMDV 2C Expression Vector
[0095] The genome sequence of FMDV 2C protein was retrieved from NCBI GenBank data (GI: 5921457, O strain Chu-Pei) and oligonucleotides for the synthesis of whole 2C gene and sequencing were synthesized at ResGen (Huntsville, Ala.). The coding DNA sequence is 954 nucleotides long, which encodes 318 amino acids (SEQ ID NO: 119).
[0096] (i) Construction of Synthetic Full-Length 2C Gene
[0097] To obtain the 2C gene of FMD virus, 24 oligonucleotide primers were synthesized, each with complementary ends, at Resgen.
[0098] We used the recursive PCR method to assemble the oligonucleotides into full 2C gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers. Because of the ...
example 3
[0107] Preparation of Recombinant FMDV 3ABC Antigen
[0108] A. Construction of FMDV 3ABC Expression Vector
[0109] The genome sequence of FMDV 3ABC protein was retrieved from NCBI GenBank data (GI: 5921457, O strain Chu-Pei) and oligonucleotides for the synthesis of whole 3ABC gene and sequencing were synthesized at ResGen (Huntsville, Ala.). The coding DNA sequence is 1281 nucleotides long, which encodes 427 amino acids (SEQ ID NO: 120).
[0110] (i) Construction of Synthetic Full-Length 3ABC Genes
[0111] To obtain the 3ABC gene of FMD virus, 33 oligonucleotide primers were synthesized, each with complementary ends, at Resgen.
[0112] We used the recursive PCR method to assemble the oligonucleotides into full 3ABC gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers....
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