Method for prokaryotic expression of recombinant chicken angiopoietin-like protein 4 and application thereof

A prokaryotic expression and protein technology is applied in the field of prokaryotic expression of recombinant chicken angiopoietin-like protein 4, which can solve the problems of unsuccessful expression of recombinant chicken ANGPTL4 protein and low sequence homology of ANGPTL4 protein.

Pending Publication Date: 2019-12-13
LINYI UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There have been recombinant ANGPTL4 protein products expressed by the eukaryotic expression system (HEK293) on mice and humans, but the homology of the ANGPTL4 protein sequence on chickens and other species is low. The inventors tried to use the eukaryotic expression system (HEK293) to express recombinant chicken ANGPTL4, but it was not successful, and there is no report on the successful expression of recombinant chicken ANGPTL4 protein

Method used

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  • Method for prokaryotic expression of recombinant chicken angiopoietin-like protein 4 and application thereof
  • Method for prokaryotic expression of recombinant chicken angiopoietin-like protein 4 and application thereof
  • Method for prokaryotic expression of recombinant chicken angiopoietin-like protein 4 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the preparation of recombinant chicken ANGPTL4 protein

[0058] Use UniProt to find the amino acid sequence of chicken ANGPTL4 (serial number F1NUQ4) and the signal peptide part (1-18 amino acids), remove the signal peptide and add the His-SUMO tag to the N-terminus, compare the preferred codons of E. coli, according to the codons On the basis of not changing the amino acid composition and arrangement sequence, the gene sequence encoding His-SUMO-ANGPTL4 was transformed and replaced with codons preferred by Escherichia coli to obtain a new gene sequence encoding His-SUMO-ANGPTL4 (SEQ ID NO.2), its protein amino acid sequence is SEQ ID NO.4, sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for whole gene synthesis, and constructed into pET21a with Nde I and Xho I restriction sites ( +) in the expression vector.

[0059] 2. Transformation of expression strains

[0060] The recombinant prokaryotic expression plasmid pET21a-His-SUMO-ANGPTL4 was transformed...

Embodiment 2

[0077] Example 2, Effects of different concentrations of recombinant chicken ANGPTL4 protein on broiler blood biochemical indicators

[0078] 1. Experimental design

[0079] Thirty-six AA meat roosters with similar body weight at 35 days of age were selected in the fasting state (fasting for 12 hours), and were randomly divided into 6 treatments, with 6 repetitions for each treatment, and 1 chicken for each repetition. Each group was treated with intravenous injection of 0, 20, 100, 500, 2500 and 12500 ng / kg of recombinant chicken ANGPTL4 protein according to the body weight of the broiler, and the fasting blood of the test broiler was collected 30 minutes after the injection, the serum was separated, stored at -20°C, and measured by an automatic biochemical analyzer Blood biochemical indicators such as triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL), and the content of very low-density lipoprotein (VLDL) by enzyme-li...

Embodiment 3

[0088] Embodiment 3, the influence of recombinant chicken ANGPTL4 protein on fat metabolism of broiler skeletal muscle

[0089] How to increase the fat content of broiler skeletal muscle in a targeted manner is a major scientific problem in the current poultry industry. Studies have found that the addition of Clostridium butyricum can achieve this goal, but its molecular mechanism has not yet been clarified. ANGPTL4 is a protein closely related to animal skeletal muscle fat metabolism, and is an important medium connecting intestinal microorganisms and host skeletal muscle fat metabolism. The previous inventor's animal experiments found that Clostridium butyricum can cause ANGPTL4 in the ileum and serum of broiler chickens. Therefore, it is speculated that ANGPTL4 plays an important role in the directional increase of fat content in broiler skeletal muscle by Clostridium butyricum. In order to further confirm this hypothesis, this experiment partly takes prokaryotic expression...

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Abstract

The invention provides a method for prokaryotic expression of recombinant chicken ANGPTL4 protein, comprising the following steps: (1) artificially synthesizing a gene sequence of recombinant chickenANGPTL4, and constructing the gene sequence into a prokaryotic expression vector to construct a recombinant prokaryotic expression vector; (2) transforming Escherichia coli E.coli competent cells by using the recombinant prokaryotic expression vector, and screening positive bacterial colonies; (3) carrying out amplification culture on the recombinant positive plasmid single colony, and adding an inducer to carry out inducible expression; (4) carrying out inclusion body renaturation by adopting a dilution renaturation method; and (5) purifying the inclusion body renaturation protein. By constructing a prokaryotic expression vector of the recombinant chicken ANGPTL4, the recombinant chicken ANGPTL4 is efficiently expressed in escherichia coli, the expressed recombinant protein exists in theform of an inclusion body, correct folding of the protein is promoted through inclusion body renaturation and renaturation protein purification, and the expression quantity of the fusion protein is increased. The recombinant chicken ANGPTL4 protein provided by the invention can adjust fat metabolism of broiler chickens, and provides a good test material for development of chicken ANGPTL4 functionresearches in the future.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application of prokaryotic expression of recombinant chicken angiopoietin-like protein 4. Background technique [0002] With the widespread use of feed additives and the continuous improvement of feed nutrition levels, the fat deposition ability of broilers is increasing day by day. However, the media and their mechanisms of action linking nutritional factors and fat metabolism of broilers have not yet been clarified. Angiopoietin-like protein 4 (angiopoietin-like protein 4, ANGPTL4), also known as PPAR angiopoietin-related protein and fasting-inducing factor, is a kind of secreted protein with high biological activity, which can be affected by factors such as microorganisms and fasting. secretion. In recent years, studies have found that when the conditioned medium with differences in ANGPTL4 is used to culture the human liver cancer cell line HepG2, the expression of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/515C07K19/00A61D7/00C12R1/19
CPCC12N15/70C07K14/515A61D7/00C07K2319/21
Inventor 赵旭黄华山苏闪闪
Owner LINYI UNIVERSITY
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