Recombinant alpha protein for inhibiting clostridium perfringens infection and preparation method and application thereof
A technology of α protein and protein, which is applied in the field of recombinant α protein for inhibiting Clostridium perfringens infection and its preparation and application, which can solve the problems such as difficulty in increasing the renaturation rate
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Embodiment 1
[0089] Example 1. Soluble expression of α-hisY
[0090] 1. Synthetic genes
[0091] The present application designed three kinds of recombinant α genes, namely α-hisY gene shown in SEQ ID No.1, α-hisW gene shown in SEQ ID No.3, and pmα-hisW gene shown in SEQ ID No.4.
[0092] Both the α-hisY gene and the α-hisW gene encode the protein α-his shown in SEQ ID No.2. The pmα-hisW gene encodes the protein pmα-hisW shown in SEQ ID No.5. α-his is a protein obtained by deleting amino acid residues 52-146 of pmα-hisW.
[0093] The α-Y gene shown in the 151-1062 position of SEQ ID No.1 (coding the protein shown in the 51-353 amino acid residues of SEQ ID No.2) was synthesized by chemical synthesis, SEQ ID No. .3 the α-W gene shown in No. 151-1062 (the protein shown in the No. 51-353 amino acid residues of encoding SEQ ID No.2), shown in No. 151-1347 of SEQ ID No.4 pmα-W gene (encodes protein pmα-W represented by amino acid residues 51-448 of SEQ ID No.5).
[0094] 2. Construction of...
Embodiment 2
[0114] Embodiment 2, animal immunoprotective test of α-his
[0115] 1. Preparation of anti-Clostridium perfringens vaccine
[0116] The α-his protein purified by molecular sieves in Example 1 was dissolved in sterile PBS to obtain an α-his solution with an α-his concentration of 1000 μg / mL for immunization. The α-his solution and Freund's adjuvant were mixed in an equal volume of 1:1, and emulsified to prepare an oil emulsion vaccine, which was named the first vaccine. The α-his solution and incomplete Freund's adjuvant were mixed in an equal volume of 1:1, and emulsified to prepare an oil emulsion vaccine, which was named the second-immunity vaccine.
[0117] Take out the A-type Clostridium perfringens virulent strain C57-10, the B-type Clostridium perfringens virulent strain C58-5, and the C-type Clostridium perfringens virulent strain purchased from the China Veterinary Drug Administration. Strain C59-4, Clostridium perfringens type D virulent strain C60-11. In the ultra...
Embodiment 3
[0140] Example 3, Optimization of α-his Induced Expression Conditions
[0141] 1. Optimization of induction temperature and time
[0142] Inoculate BL21(DE3) / pET30a-α-Y in LB liquid medium containing 50 μg / ml kanamycin (add kanamycin to LB liquid medium until the concentration of kanamycin is 50 μg / ml to obtain culture medium) at 37°C, using a Thermo MaxQ6000 full-temperature shaker at 200rpm to shake and cultivate to OD 600 When the value (the LB liquid medium containing 50 μg / ml kanamycin was used as the blank control) reached 0.6, isopropylthio-β-D-galactoside (IPTG) was added to induce the following six kinds of expression respectively. The first induced expression was induced with 0.75 mM IPTG for 1 hour at 37°C. The second induced expression was induced with 0.75 mM IPTG for 2 hours at 37°C. The third induced expression was induced with 0.75 mM IPTG for 4 hours at 37°C. The fourth induced expression was induced with 0.75 mM IPTG for 5 hours at 37°C. The fifth induce...
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