Recombinant pig growth hormone and preparation method and application thereof

A growth hormone and recombinant plasmid technology, applied in the direction of growth hormone, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of short half-life, and achieve the effect of simple method, short half-life and low cost

Active Publication Date: 2021-01-05
安徽中起生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, some relevant institutions have developed a fusion protein of porcine growth hormone, which overcomes the problem of gastrointestinal absorption, but does not solve the problem of short half-life

Method used

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  • Recombinant pig growth hormone and preparation method and application thereof
  • Recombinant pig growth hormone and preparation method and application thereof
  • Recombinant pig growth hormone and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027]Example 1: Polynucleotide sequence

[0028]A polynucleotide sequence includes a guide sequence that is sequentially connected and three repeated full-length sequences of porcine growth hormone encoding genes. The full-length sequences of the porcine growth hormone encoding genes are connected by a flexible linker sequence.

[0029]Among them, the full-length sequence of the porcine growth hormone encoding gene refers to GENBANK accession number X53325.1, the specific sequence is shown in SEQ ID NO. 1, the guide sequence is shown in SEQ ID NO. 2, and the flexible linker sequence is shown in SEQ ID NO. 3. Shown.

Embodiment 2

[0030]Example 2: Preparation of recombinant porcine growth hormone

[0031](1) Obtain the gene sequence of recombinant porcine growth hormone:

[0032]Refer to GENBANK accession number X53325.1, take the full-length porcine growth hormone sequence, make three repeats, add the restriction site EcoRI and guide sequence upstream, and use a flexible linker between the three repeats to protect the spatial structure of the target fragment. Add restriction site Not I and protective base downstream to obtain as shown in SEQ ID NO.4.

[0033]The target gene sequence was sent to BGI for gene synthesis, and the sequence was qualified to obtain the target gene sequence.

[0034](2) Construction of engineering bacteria:

[0035]Insert the polynucleotide sequence in Example 1 into the Pichia pastoris expression vector to obtain the pPICZαA plasmid, and then use the two endonucleases EcoR I and Not I to double digest the synthesized gene and pPICZαA plasmid, and double digest The product was ligated with ligase ...

Embodiment 3

[0040]Example 3: Detection of serum concentration after oral administration of recombinant Sanlian porcine growth hormone

[0041]Three healthy male SD rats (purchased from Anhui Experimental Animal Center) were selected, weighing about 200 grams. After fasting without water for 12 hours, the recombinant porcine growth hormone PBS prepared in Example 2 was diluted to 1 mg / ml, and each rat was given 1 mg of recombinant triple porcine growth hormone by gavage. Take 200μL of blood from the orbit before the start of the experiment (0h) and 1, 2, 4, 8, 12, 18, 24h after gavage, place it at 4℃ for 1h, centrifuge at 7000rpm to separate the serum, and freeze it immediately at -70℃, the last time After blood collection, it was also frozen and stored at -70°C for more than 2 hours to ensure that all serums were frozen and thawed once during the ELISA test. Operate according to Biorbyt (orb439756) instruction to determine the content of porcine growth hormone in rat serum. It can be detected in t...

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Abstract

The invention discloses a recombinant pig growth hormone and a preparation method and application thereof. The preparation method comprises the following steps of: inserting a polynucleotide sequencecontaining a plurality of pig growth hormone full-length sequences into a pichia pastoris expression vector to obtain a recombinant plasmid; introducing the recombinant plasmid into a pichia pastoriscompetent cell to construct a recombinant engineering bacterium; and performing fermentation expression and protein purification on the recombinant engineering bacterium to obtain a target protein, namely the recombinant pig growth hormone. The recombinant pig growth hormone can be applied as a pig feed additive. The recombinant pig growth hormone has the advantages that the protein molecular weight is increased by adopting a multi-linkage mode, so that the problem that the hormone is digested in the stomach and loses functions is solved; meanwhile, a plurality of repeated fragments exist, sothat the problem of short half-life period can be well solved; and in addition, the recombinant pig growth hormone is produced by adopting a recombination mode, is simpler and lower in cost compared with an extraction method, and can be widely applied as the feed additive.

Description

Technical field[0001]The invention relates to the technical field of gene recombination, in particular to a recombinant porcine growth hormone and its preparation method and application.Background technique[0002]Recombinant protein is a protein obtained by using recombinant DNA or recombinant RNA technology. The production of recombinant proteins in vitro is mainly divided into two types: prokaryotic expression and eukaryotic expression: prokaryotic expression systems are prokaryotic expression systems; eukaryotic expression systems are further divided into: yeast expression system, insect cell expression system and mammalian cell expression system .[0003]Yeast expression system: It has the advantages of both prokaryotic and advanced eukaryotic systems. Yeast is a single-celled lower eukaryote. It has common culture conditions, rapid growth and reproduction, and can tolerate high hydrostatic pressure. When it is used to express genetic engineering products, it can be produced on a l...

Claims

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Application Information

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IPC IPC(8): C12N15/18C12N15/81C07K14/61A23K20/184A23K50/30
CPCC07K14/61C12N15/815A23K20/184A23K50/30
Inventor 卞传忠
Owner 安徽中起生物科技有限公司
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