Cloning expression and application of fusion bursin

A technology of tripeptide bursin and fusion peptide, which is applied in the field of molecular biology, can solve the problems of finding ALV strains and the loss of breeding industry, etc., and achieve good market application prospects, strong inhibition of retroviruses, and good immune enhancement The effect of action

Inactive Publication Date: 2019-09-10
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Safe and effective biological products (such as vaccines) have not been developed to prevent AL. The main reason is that no non-pathogenic ALV strains can be found, which brings severe challenges to veterinary workers and causes breeding huge loss to the industry

Method used

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  • Cloning expression and application of fusion bursin
  • Cloning expression and application of fusion bursin
  • Cloning expression and application of fusion bursin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Cloning expression of fusion tripeptide bursin

[0025] 1. Involvement and synthesis of primers

[0026] According to the specificity of foreign protein encoded by Escherichia coli, a tandem tetrapeptide BS (8) was designed and synthesized, and a fusion peptide sequence was added at the end. The amino acid sequence of the fusion BS was GSPRM KNPY KNPY KNPY KNPY KNPY KNPY KNPY KNPYHMGGGG SKLQRLMEDICLPRWGCLWEDDF, the core of which was The nucleotide sequence is: ATGAA AAACC CGTAT AAAAA CCCGTATAAA AACCC GTATA AAAAC CCGTA TAAAA ACCCG TATAA AAACC CGTAT AAAAA CCCGT ATAAAAACCC GTAT C ATAT G GGCGG CGGCG GCAGC AAGCT T CAGC GCCTG ATGGA AGATA TTTGC CTGCCGCGTT GGGGC TGCCT GTGGG AAGAT GATTT TTAAT AA was sent to Shanghai Sangon Bioengineering Co., Ltd. for synthesis and cloned into the pET-32a plasmid to construct the recombinant plasmid pET-BS. In addition, according to the published sequence of pET-32a and the designed and synthesized recombinant pET-BS, specific pri...

Embodiment 2

[0039] Example 2 Purification of Soluble Protein and BCA Quantification

[0040] According to the purification kit ( Protein Iso TM Ni-NTA Resin) instructions. The specific purification steps are as follows:

[0041] (1) Cleaning of the chromatographic column: first use ultrapure water to rinse the chromatographic column so that the negative of the chromatographic column is close to the bottom of the column.

[0042] (2) Column packing: Resuspend the medium, add an appropriate amount of medium to the chromatography column according to the amount of BS protein to be purified, let it stand, and discard the effluent.

[0043] (3) Equilibration: Equilibrate the column with 8 times the volume of the column packing medium in soluble equilibration buffer (containing 10 mM imidazole), and discard the effluent.

[0044] (4) Sample loading: Slowly add the BS soluble protein after high-speed centrifugation into the chromatography column, and collect the effluent.

[0045] (5) Washin...

Embodiment 3

[0050] Example 3 Establishment of an animal model of ALV congenital infection

[0051] (1) Culture of ALV and titration of virus infectivity (TCID50)

[0052] The ALV-FJ15HT0 strain was inoculated on a monolayer of DF-1 cells, placed in an incubator containing 5% CO2 at 37 °C, and collected after one week. First, it was frozen and thawed three times in a row, then filtered with a 0.22 μm filter, and finally frozen. Store in a -80°C freezer. The determination of ALV TCID50 selects the ALV p27 antigen detection kit produced by IDEXX Company. The assay was performed according to the kit instructions.

[0053] (2) Incubation of chicken embryos

[0054] The purchased SFP chicken embryos were placed in a fully automatic incubator. The specific incubation temperature and humidity for different embryo ages are shown in Table 3:

[0055] Table 3 Conditions for incubation of chicken embryos

[0056]

[0057] (3) SPF chicken embryo yolk sac inoculation

[0058] At the 7th embryo...

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Abstract

Soluble fusion bursin is designed and subjected to cloning expression. The soluble fusion bursin serves as an immune adjuvant for animals after being purified, a good immunoenhancement effect and a growth promotion function are embodied in a chicken group with an ALV infection background, and the soluble fusion bursin has a relatively strong effect of inhibiting reverse transcription viruses in vivo and in vitro and has good market application prospects.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to the cloning, expression and application of fusion tripeptide bursin. Background technique [0002] Tripeptide bursin (bursin, BS), also known as bursin peptide and bursal active peptide, is a small molecular peptide active substance present in the poultry bursa. Its structure is lysine-histidine-glycine (Lys-His-Gly-NH2). Research results at home and abroad show that BS has the function of enhancing immunity and promoting the growth of livestock and poultry. BS can increase the content of the second messenger cGMP in serum, increase the ratio of cGMP and cAMP, increase the speed of DNA transcription into mRNA in B cells, and promote the synthesis of proteins in B cells, thereby increasing the level of specific antibodies in serum. [0003] In recent years, the prevalence of avian leukosis (Avian Leukosis, AL) has gradually expanded, causing huge economic losses. AL...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/39A61P37/04
CPCA61K39/39A61K2039/55516A61P37/04C07K14/00C07K2319/00
Inventor 吴晓平余深翼曾宇坤赵金荣卢荣彬安亚娟关文超龚祖新周东辉吴异健
Owner FUJIAN AGRI & FORESTRY UNIV
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