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Hippocampus antibiotic peptide pichia pastoris engineering strain and use thereof in breeding

A technology of Pichia pastoris and Pichia pastoris, applied in the field of genetic engineering, can solve problems such as influence, and achieve the effects of prolonging storage period, good water solubility and good stability

Inactive Publication Date: 2011-03-23
广东中大南海海洋生物技术工程中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the abuse of antibiotics in the world is serious. Some scholars have worriedly proposed that because human beings are always in a passive and backward situation in the "arms race" with bacteria, when one day the most powerful antibiotics developed by humans will be due to superbugs What will the world be like when the existence of the world becomes invalid? According to statistics, there are currently as many as 20 kinds of antibiotics used as feed additives for humans and animals or for livestock and poultry in China. With my country's entry into WTO, antibiotics as feed additives will seriously affect the competitiveness of our livestock products in the international market

Method used

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  • Hippocampus antibiotic peptide pichia pastoris engineering strain and use thereof in breeding
  • Hippocampus antibiotic peptide pichia pastoris engineering strain and use thereof in breeding
  • Hippocampus antibiotic peptide pichia pastoris engineering strain and use thereof in breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 The production method of recombinant yeast expression antimicrobial peptide HKPLP product

[0019] (1) Synthesis of antimicrobial peptide gene hkplp

[0020] The antimicrobial peptide gene hkplp was optimally designed according to yeast preferred codons, and two primers, PLP1 and PLP2, were synthesized respectively. There was a 15 bp complementary paired sequence between the two primers, which were used as templates for PCR amplification. Synthetic antimicrobial peptide antimicrobial peptide gene hkplp flow chart is as follows figure 1 shown.

[0021] PLP1:

[0022] 5'-CGGGATCCACCATGTTTTTGGGACTTATTTTTCACGGTCTGGTTCACGCCGGAAAGCTG-3'

[0023] PLP2:

[0024] 5'-CGGAATTCTCATTAACCACGATTTCTGTGGATCAAACCGTGGATCAGCTTTCCGGCGTG-3'

[0025] Reaction conditions:

[0026] 10×PCR reaction buffer 2μL

[0027] dNTPs (2.5mmol / L) 2μL

[0028] PLP1 1 μL

[0029] PLP2 1 μL

[0030] pfu Taq (2U / μL) 0.5μL

[0031] ddH2O 13.5μL

[0032] ...

Embodiment 2

[0058] Example 2 Application of recombinant antimicrobial peptide HKPLP to promote the growth of prawns

[0059] The antimicrobial peptide product is used as a feed additive to make feed for raising prawns. There are three groups of experimental components, which are low, medium and high antimicrobial peptide groups, and the final concentrations are respectively added to 200ppm, 500ppm and 1000ppm. Three treatments are established in each experimental group. , each treatment prawn initial tail number is 30, control the same water quality, temperature and dissolved oxygen of each experimental group, respectively record the survival rate of each treatment, the average weight of each tail at the beginning and end, and the food intake of a single tail, the results are shown in Table 2 below, It can be seen from the results that the average survival rate and single-tail weight gain rate of the experimental group added with antimicrobial peptide feed additives were significantly high...

Embodiment 3

[0062] Example 3 Application of recombinant antimicrobial peptide HKPLP to promote chicken growth

[0063] Select 1000 freshly hatched commercial AA broilers with the same male-to-female ratio and divide them into a control group and an experimental group, each with 500 chickens. The control group selected the full-price feed prepared with 4% broiler premix, and added antibiotics according to the recommended amount. The test group did not add antibiotics, and the feed was full-price feed prepared by 4% broiler premix, and 0.04% antimicrobial peptide HKPLP was added, and the test period was 6 weeks. The feeding conditions of each group were the same, and the feeding management was carried out according to the conventional feeding management system of the chicken farm.

[0064] At the beginning of the test, the weight of the chickens in each group was weighed, and the body weight and feed weight of the chickens were weighed on an empty stomach in the morning on the 21st and 42n...

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Abstract

The invention relates to a natural antibiotic peptide HKPLP derived from hippocampus and a gene hkplp for coding the protein. The DNA sequence of the natural antibiotic peptide HKPLP is represented by the sequence No.1 in a sequence table, and the protein coded by the gene (HKPLP) is represented by the sequence No.2 in the sequence table. In the invention, a new hkplp antibiotic peptide gene is designed according to the preferred codons of the pichia pastoris, the DNA sequence of the gene is synthesized and transferred to the pichia pastoris KM71 to be expressed, and thus, the pichia pastoris engineering strain of the antibiotic peptide is formed. Meanwhile, the fermentation conditions of the strain are optimized to realize high-density fermentation and high-efficiency expression. The fermentation liquor of the engineering strain has obvious inhibition effects on Gram positive bacteria, Gram negative bacterial, fungi and the like. The processed product of the fermentation liquor was added into feed for shrimp, chicken and the like, and good experimental effects were obtained. The antibiotic peptide product can replace antibiotic, prevent diseases of the digestive tract, promote growth, increase weight and used as an additive for livestock and aquatic product feed.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an antibacterial peptide gene transformation Pichia engineering bacteria and its preparation method and application. Background technique [0002] The seahorse (Hippocampus) is a peculiar marine creature, belonging to the subphylum Vertebrate, the class Fish, the order Syngnathidae, and the order Syngnathidae. At present, there are 35 species of seahorses identified in the world. In my country, seahorses mainly inhabit the southeastern waters, and there are five common species (Zhang Zhaohui et al., 1998), namely, the lined seahorse (H. , zebra seahorse (H.trimaculatus) and Japanese seahorse (H.japonicus). [0003] Antimicrobial peptide is an important part of the immune defense system in animals. It is a short-chain amino acid peptide and is known as a pollution-free natural antibiotic. It has broad-spectrum antibacterial, no drug resistance, heat stability, good water solubi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P21/02A23K1/17C12R1/84A23K20/195
Inventor 徐安龙荆琛峰李本涛吴松青王瑞黄海林徐梁洪黄赛鸳梁东
Owner 广东中大南海海洋生物技术工程中心有限公司
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