Tibetan sheep myostatin recombinant expression protein

A technology of hiding sheep and protein, applied in the field of genetic engineering, can solve problems such as overexpression of propeptide, lower survival rate of calves, and impact on animal heart load

Inactive Publication Date: 2011-12-21
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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AI Technical Summary

Problems solved by technology

Mutations in the MSTN gene lead to increased body weight and increased feed conversion rate, but the unfavorable effects are decreased fertility of cows, decreased survival rate of calves and delayed sexual maturity, as well as loss of intermuscular fat and connective tissue alkali less, weight loss of other organs, etc.
It can be seen that the application of gene knockout, gene targeting, transgenic and other technologies to destroy the structure and activity of MSTN in the body can certainly promote muscle growth, but at the same time it will inevitably affect the animal's heart load, reproductive ability, etc.
And at present, there has been a lot of controversy about the safety of genetically modified foods, so the food safety of the obtained animal muscle cannot be guaranteed.
At present, the expression system of Escherichia coli is most commonly used for recombinant expression of MSTN. Although this system has the advantages of clear genetic background, fast reproduction, low cost, high expression level, and easy operation, it is relatively inferior to the mammalian cells distributed by MSTN. As far as Escherichia coli is concerned, the expression system of Escherichia coli is still relatively low-level, and cannot fully realize the advanced assembly of recombinant proteins and the formation of complex spatial conformations, so most of the expression products are inclusion bodies with poor solubility. Another important factor for the low price
In addition, studies have shown that direct application of the full-length MSTN-encoding gene during expression and purification can easily lead to overexpression of the propeptide, and the N-terminal propeptide and C-terminal mature peptide of mice with overexpression of the propeptide cannot be separated normally, resulting in no biological activity. polymer

Method used

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  • Tibetan sheep myostatin recombinant expression protein
  • Tibetan sheep myostatin recombinant expression protein
  • Tibetan sheep myostatin recombinant expression protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1, construction of Tibetan sheep pET28a(+)-MSTN' recombinant vector

[0115] 1. Primer design

[0116] According to the sheep myostatin gene sequence (AF019622), use Vector NTI software to design myostatin coding gene primers, and the primer sequences are as follows:

[0117] Upstream primer P1: 5′-ATGCAAAAACTGCAA-3′

[0118] Downstream primer P2: 5′-TCATGAGCACCCCACAGCGATCTACT-3′

[0119] 2. RT-PCR amplification of Tibetan sheep myostatin fragment

[0120] (1) RNA extraction from Tibetan sheep leg skeletal muscle

[0121] According to the total RNA extraction method provided by Invotrogen, the total RNA of Tibetan sheep leg skeletal muscle was extracted according to the following steps:

[0122] ①Take 50-100 mg of Tibetan sheep leg skeletal muscle tissue stored at -70°C, shred it, transfer it to a 20 mL homogenizer with 1 mL Trizol added in advance, and homogenize until the cells are completely lysed;

[0123] ② Transfer the homogenized liquid into a 2mL ce...

Embodiment 2

[0196] Example 2, expression of Tibetan sheep pET28a(+)-MSTN'-E.coli BL21(DE3) genetic engineering bacteria

[0197] 1. Construction of Tibetan Sheep pET28a(+)-MSTN′-E.coli BL21(DE3) Genetic Engineering Bacteria

[0198] The pET28a(+)-MSTN' recombinant vector was transformed into Escherichia coli BL21(DE3) host bacteria, and the specific operation steps were the same as the transformation operation in the Sanzang sheep myostatin clone in step 1 of the above-mentioned Example.

[0199] 2. IPTG induces the expression of pET28a(+)-MSTN'-E.coli BL21(DE3) genetic engineering bacteria

[0200] (1) Pick a single recombinant bacterium and inoculate it in LB medium, culture overnight at 37°C and 120rpm with shaking, the volume ratio of genetically engineered bacteria solution to LB medium is 1:100, and use the bacterial sample containing empty vector as a control ;

[0201] (2) The next day, the activated seed bacteria of the experimental group and the control group were inoculated i...

Embodiment 3

[0209] Example 3, Tibetan sheep MSTN mature peptide protein purification

[0210] 1. Inclusion body isolation

[0211] (1) The expression bacteria induced by IPTG were centrifuged at 12000r / min at 4°C for 15min to collect the bacteria;

[0212] (2) Add PBS buffer solution to the cells, add 10 mL of wet weight of 1 g cells, resuspend the cells, and wash twice;

[0213] (3) The bacterial cell suspension was placed on ice for 20 minutes and ultrasonically crushed, broken for 5 seconds, interrupted for 9 seconds, centrifuged at 12000 r / min at 4°C for 15 minutes, and the precipitate was collected;

[0214] (4) Add PBS buffer containing 0.1% Triton X-100 to resuspend the pellet, add 20 mL of buffer to the wet weight of 1 g of bacteria, let stand at 4°C for 20 min, and wash repeatedly 5 times;

[0215] (5) Wash the inclusion body 5 times with PBS buffer containing 2M urea in the same way, centrifuge at 12000r / min at 4°C for 15min, and collect the precipitate;

[0216] (6) The wash...

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Abstract

The invention discloses a recombinant Tibetan sheep myostatin protein. In the invention, a Tibetan sheep myostatin (MSTN) coding gene is cloned, the gene is used as a template for polymerase chain reaction (PCR) amplification, the bioactive region of the MSTN coding gene is cloned, a recombinant expression vector is constructed, the cloned bioactive region is transferred into Escherichia coli to construct gene engineering bacteria, and the gene engineering bacteria are subjected to isopropyl thiogalactoside (IPTG) induced expression and purification of MSTN protein. Then the MSTN protein is used to actively immunize a domestic rabbit to prepare polyclonal antibody serum, and an indirect enzyme-linked immuno sorbent assay (ELISA) is used to detect the immunogenicity of the MSTN protein. The recombinant Tibetan sheep MSTN protein is used to immunize a mouse actively so as to detect the influence of the recombinant MSTN protein on the growth of the mouse. The purity and immunogenicity of the recombinant Tibetan sheep MSTN protein provided by the invention are high, and the actively immunizing MSTN protein can obviously increase the diameter and area of muscle fibers of the mouse and promote the muscle fibers to increase. Thus, the recombinant Tibetan sheep MSTN protein can be used for active immunizing of Tibetan sheep and help to accelerate the growth of Tibetan sheep and to improve meat performance.

Description

Technical field [0001] The present invention involves the field of genetic engineering technology. Specifically, it involves a type of genetic engineering bacteria, reorganized protein expression, preparation of polyxolite and immune detectionTechnology. Background technique [0002] Tibetan sheep is a unique lamb species formed by long -term natural breeding in the high -cold ecological environment. It has the characteristics of cold resistance, thick feeding, and strong disease resistance.The advantageous animal species is also an important means of production and living materials for the people of pastoral areas. In 2000, it was included in the Ministry of Agriculture in the "National -level Live and Poultry Variety Resources Protection List"."Tibetan cotton mutton is rich in nutrition, tender and soft muscle fiber, high protein content, rich in histidine, arginine, suline, alanine, glycine, proline and other amino acids.It is favored by consumers and has huge market potential...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/12C12N15/66C12N1/21C07K14/475C07K1/16A01K67/02C12R1/19
Inventor 徐亚欧毛亮郑玉才
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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