Recombinant alpha protein for inhibiting Clostridium perfringens infection and its preparation method and application
A technology for Clostridium perfringens and alpha protein, which is applied in the directions of antibacterial immunoglobulins, botanical equipment and methods, biochemical equipment and methods, etc., and can solve 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 of SEQ ID No.1 (the protein shown in the 51-353 amino acid residues of encoding SEQ ID No.2) is synthesized by chemical synthesis method, SEQ ID No. α-W gene shown in No. 151-1062 of 3 (the protein shown in No. 51-353 amino acid residues of coding SEQ ID No.2), pmα shown in No. 151-1347 of SEQ ID No.4 -W gene (encodes protein pmα-W represented by amino acid residues 51-448 of SEQ ID No.5).
[0094] 2. Construction of recombi...
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 ThermoMaxQ6000 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 induced...
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