Method for preparing TGEV (Transmissible Gastroenteritis Virus)-S gene corns
A technology of transgenic plants and corn, applied in the directions of botanical equipment and methods, pharmaceutical formulations, and medical preparations containing active ingredients, etc., can solve the problems of low mortality, large economic losses, and reduced feed rate of return.
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Embodiment 1
[0041] Example 1. Preparation and verification of gene
[0042] The TGEV-S gene was cloned from the TGEV-S recombinant plasmid virus by PCR.
[0043] The recombinant plasmid TS (the 2.2kb N-terminal fragment of the S gene containing TGEV was connected with the pMD18-T plasmid vector) was provided by Mr. Zhang Li from the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry; upstream primers (TGEVS-F, 5`-GGA) were used. TCC ATG AAA AAA CTA TTT GTG G-3') and downstream primers (TGEVS-R, 5'-GAG CTC ATT ATA CAT CAC ATG GCG TTA CAG-3'), the S gene was cloned from the recombinant plasmid TS by PCR, And Shanghai Yingjun Biotechnology Co., Ltd. for sequencing. Reaction system: 10×PCR Buffer 2.5 μL, upstream and downstream primers 1 μL each, dNTP (2.5 mM) 2 μL, template 0.5 μL, Ex Taq DNA polymerase 0.5 μL, ddH2O 17.5 μL, total volume 25 μL. The reaction procedure was as follows: pre-denaturation at 95 °C for 5 min; denaturation at 94 °C ...
Embodiment 2
[0046] Example 2. Preparation of transgenic plants
[0047] 1. Recombinant expression vector pBAC176
[0048] The base vector pSP72 was purchased from Promega, catalog number P2191.
[0049] Agrobacterium nopaline synthase gene NOS 3′-terminal terminator (0.26kb) sequence was recovered from pBI221 (Genbank ACCESSION AF502128, from positions 2778-3030) with SacI and EcoRI double digestion, and inserted into the Sad and EcoRI sites of pSP72 (Promega) , the plasmid pBPC18 (2.7kb) was obtained by the blue-white screening method.
[0050] E35S promoter (Enhanced CaMV 35S) (Seq ID: No. 3) and Hsp70 intron1 (Seq ID: No. 4) were both artificially synthesized sequences, synthesized by Shanghai Sangong Company. The E35S promoter was digested with HindIII / XbaI and inserted into the HindIII / XbaI site of pBPC18 to obtain an intermediate recombinant vector; Hsp70 intron1 was inserted into the BamHI site of the intermediate recombinant vector with BglII / BamHI enzyme digestion, and the resu...
Embodiment 3
[0081] Example 3. Transgenic maize immunized animals produce antibodies
[0082] 1. Vaccine preparation and immunization dose
[0083] Take 0.5 g of the transgenic maize leaves prepared in Example 2 or 0.5 g of non-transgenic maize leaves and grind in liquid nitrogen, add PBS (pH=7.4) at 1:4 (W / V) and mix well. Centrifuge at 12000rpm and 4°C for 10min, take the supernatant and repeat once. The supernatant and adjuvant were mixed and emulsified at a ratio of 1:1 (complete Freund's adjuvant was added for the first dose, and incomplete Freund's adjuvant was added to the others).
[0084] For the first immunization, each mouse was injected with 0.2ml (equivalent to 50mg of fresh leaf weight), and the same dose was also used for booster immunization.
[0085] 2. Immunization of experimental animals
[0086] The 4-week-old female BALB / c mice were randomly divided into five groups (5 mice / group), and the emulsified vaccine was subcutaneously injected into the mice, and immunized o...
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