Antioxidant polypeptide expression pichia pastoris engineering bacteria and application

[0049] A Pichia pastoris KM71H FZM expressing the antioxidant polypeptide VECYGPNRPQF 2014 AOP construction, including the following steps:

[0050] 1 Material:

[0051] 1.1 Strains and vectors

[0052] See Table 1 for strains and vector genotypes and sources.

[0053] Table 1 Strains and vector genotypes and sources

[0054]

[0055] 1.2 Tool enzymes:

[0056] RNaseA and Proteinase K were purchased from Shanghai Shenggong Company; restriction enzymes were purchased from Promega; 2×PCR master Mix was purchased from Beijing Tiangen Biotechnology Company.

[0057] 1.3, plasmid and yeast genome kits:

[0058] Small, medium and large extraction plasmid kits, and yeast genome kits are products of QIAGEN.

[0059] 1.4 Medium:

[0060] LB liquid medium: 5g of yeast extract; 10g of peptone; 5g of NaCl; add water to make up to 1L. LB solid medium: add agar powder at a final concentration of 1.5% to the LB liquid medium.

[0061] The following is based on figure 1 The present in

[0068] The preparation method of antioxidant polypeptide VECYGPNRPQF comprises the following steps:

[0069] A, material and solution preparation: fermentation medium, its composition is glucose 4% (m / v), ammoniacal liquor 150 μ L (commercially available, ammoniacal liquor concentration is 25%), KH 2 PO 4 0.7% (m / v), MgSO 4 .7H 2 O 0.03% (m / v), FeSO 4 .7H 2 O0.05% (m / v), MnSO 4 .H 2 O 0.05% (m / v), peptone 0.1% (m / v), pH5.5.

[0070] PTM1 formula is: copper sulfate 6g / L, potassium iodide 0.08g / L, manganese sulfate monohydrate 3g / L, sodium molybdate dihydrate 0.2g / L, boric acid 0.02g / L, zinc chloride 20g / L, cobalt chloride 0.5g / L, ferrous sulfate heptahydrate 65g / L, biotin 0.2g / L, sulfuric acid 5ml / L.

[0071] B. The preparation steps of antioxidant polypeptide VECYGPNRPQF are:

[0072] 1) Resuscitate Pichia pastoris KM71H FZM on a YPD plate containing 100 μg / ml zeocin 2014 AOP ;

[0073] 2) Pick a single clone from the resuscitated plat

[0081] A Pichia pastoris KM71H FZM 2014 AOP Application of expression and production of anti-oxidation polypeptide VECYGPNRPQF in preparation of animal feed additives.

[0082] Taking weaned piglets as an animal model, a specific experiment was carried out,

[0083] The specific steps are: according to the NRC standard, design the creep feed, add 0.03% (mass ratio) of the purified AOP protein prepared in Example 2, ascorbic acid (Vc), 2,6-di Weaned piglets were fed with tert-butyl-4-methylphenol (BHT), and their growth performance and oxidation-related biochemical indicators in plasma were observed, indicating that the antioxidant polypeptide VECYGPNRPQF obtained by the present invention can effectively relieve weaning stress and improve the growth of weaned piglets. Performance (results in Table 2 and image 3 ).

[0084] Table 2 Effect of antioxidant polypeptide VECYGPNRPQF on growth performance of weaned piglets

[0085]

[0086] In the same table, the same letter on th