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Porcine β2 adrenergic receptor fusion protein, coding gene and expression method thereof

A technology of epinephrine and fusion protein, which is applied in the field of genetic engineering and bioengineering, can solve unprecedented problems and achieve the effect of reducing production cost and high expression efficiency

Active Publication Date: 2015-08-19
江苏晶红生物医药科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using this expression system and using silkworm or silkworm chrysalis to express foreign genes, especially to express β 2 AR has no precedent

Method used

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  • Porcine β2 adrenergic receptor fusion protein, coding gene and expression method thereof
  • Porcine β2 adrenergic receptor fusion protein, coding gene and expression method thereof
  • Porcine β2 adrenergic receptor fusion protein, coding gene and expression method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment one, pig β 2 Artificial Synthesis of cDNA Gene of AR Fusion Protein A

[0043] According to the cDNA sequence of Sus scrofa beta-2-adrenergic receptor gene published in GenBank (GenBank accession number: AY526088.1), the start codon was removed at the 5' end of the sequence and a secretion signal suitable for insect expression system was added For the gene sequence of the peptide gp64, a 3×Flag tag gene sequence suitable for subsequent purification of the fusion protein was added before the stop codon at the 3' end of the sequence, and the gene sequence was codon-optimized according to the characteristics of the silkworm expression system. The optimized gene sequence is more suitable for silkworm expression system, which is mainly reflected in the following aspects:

[0044] 1. Codon Adaptation Index CAI (Codon Adaptation Index) of gene sequences

[0045] Depend on Figure 4-a It can be seen that before the codons are not optimized, the pig β 2 The ...

Embodiment 2

[0053] Embodiment two, baculovirus BmN-(ADRB 2 ) construction

[0054] 1. Transfer plasmid Bacmid-(ADRB 2 ) build

[0055] 5 μL pFastBac-(ADRB 2 ) plasmid was added to 100 μL of DH10Bac competent cells, and then heat-shocked in water bath at 42°C for 90 s after 30 minutes of ice bath. The product after heat shock was quickly placed on ice to cool for 3 min, then added to 900 mL of LB medium that had been incubated at 37 °C, and recovered by shaking at 37 °C and 220 rpm for 1 h. Take 100 μL of the recovered bacterial solution and spread it on the LB solid plate containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL X-gal and 40 μg / mL IPTG on 37 μg / mL IPTG. ℃ incubator inverted culture. After 48 hours, a single white colony was picked from the LB solid plate and added to the LB liquid medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline for overnight expansion. Bacmid genomic DNA was extracted and verified by ...

Embodiment 3

[0063] Embodiment three, pig β 2 Expression of AR Fusion Protein B in Bombyx mori BmN Cells

[0064] The P3 generation recombinant baculovirus BmNPV-(ADRB 2 ) to infect normally growing monolayer silkworm BmN cells and culture them at a constant temperature in a 26°C incubator. The normal BmN cells before infection with the virus showed a circular spindle-like adherent growth; while the BmN cells were infected with the P3 generation recombinant baculovirus BmNPV-(ADRB 2 ), the cells gradually stopped growing, the cells became round, and the nuclei were enlarged. After 72 h, the BmN cells lost their adherent properties and were suspended in the medium (as shown in Figure 10). The BmN cell culture medium after virus infection was collected, and the supernatant was obtained by centrifugation to obtain the P4 generation recombinant baculovirus BmNPV-(ADRB 2 ), while the cell pellet contained a large amount of expressed porcine beta of the present invention 2 AR fusion pr...

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Abstract

The invention provides a porcine beta2 epinephrine receptor fusion protein as well as an encoding gene thereof and a method for expressing the fusion protein by utilizing silkworm bioreactor. The method comprises the following steps of (1) colonizing a gene of an artificial synthesized encoded fusion protein A into a Bac-to-Bac baculoviru expression system carrier; (2) integrating the gene of the encoded fusion protein A on the transferring expression carrier obtained in the step (1) through the locus specificity transposition into baculovirus plasmid; (4) infecting the silkworm BmN cells with the baculovirus plasmid, and piercing inoculating the silkworm larva or pupae aged 1 to 5 by utilizing the obtained baculovirus; (5) expressing the fusion protein B through the infected silkworm BmN cell or the inoculated silkworm larva. The encoding gene is obtained by executing the codon optimization for the silkworm bioreactor, so that the protein expression efficiency is high, and the fusion protein is enabled to have a normal protein structure and biological activity.

Description

technical field [0001] The present invention relates to genetic engineering and bioengineering technology, in particular to a kind of pig beta 2 Adrenergic receptor fusion protein and its encoding gene and expression of porcine β using silkworm bioreactor 2 Methods of Adrenergic Receptor Fusion Proteins. Background technique [0002] beta 2 Adrenergic receptor agonists (abbreviated beta 2 agonist or beta 2 Stimulants, commonly known as clenbuterol, can promote the redistribution of nutrients in animals, and have the functions of promoting protein synthesis, reducing fat deposition, increasing lean meat rate, and reducing feed costs. They are often used as growth promoters and added to animal feeds. (V Tsatsaris et al, Obstetrics & Gynecology, 2001, 97(5):840-847). beta 2 The use of agonists can increase the lean meat rate of pigs and bring more economic value, but it will directly affect the quality of animal meat products, thus endangering the safety of human life. T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/72C12N15/12C12N15/63C12N7/01C12N15/866
CPCC07K14/72C07K2319/02C07K2319/43
Inventor 马永钱林徐春林陈晨王耀方
Owner 江苏晶红生物医药科技股份有限公司
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