Prothoracic gland hormone gene of calf of milk cow and its cloning method and use

A technique of prothymosin and cloning method, which is applied in the fields of molecular biology and biology, and can solve the problems of high price, high cost, application limitation, etc.

Inactive Publication Date: 2005-05-18
BIOLOGY INST OF SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thymosin α1 sold in the market is all artificially synthesized, and the cost is high
Tissue extraction is limited b...

Method used

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  • Prothoracic gland hormone gene of calf of milk cow and its cloning method and use
  • Prothoracic gland hormone gene of calf of milk cow and its cloning method and use
  • Prothoracic gland hormone gene of calf of milk cow and its cloning method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] 1. Collection and processing of calf thymus tissue from dairy cows

[0097] Thymus tissue was collected from a 12-day-old bull calf and stored frozen at -80°C. This calf bull is a Friesian dairy breed. Take 3 grams of thymus tissue stored in a -80°C refrigerator, cut it into pieces, place it in a sterile mortar, add sterile quartz sand for grinding, add 0.01M sterilized phosphate buffer at a ratio of 1:3 to make a cell suspension , stored in -20°C refrigerator for later use.

[0098] 2. Design of primers for reverse transcription and PCR amplification of cow prothymosin α gene

[0099] The prothymosin gene sequences of 6 humans, 1 chicken and 2 mice were downloaded from GenBank, and the accession numbers are AF348514, BC003510, BC022433, BC051265, J04799, L21693 and BM486191. Using biological software to compare and analyze the sequence differences or homology of the above genes, a pair of primers were designed, which is of great significance in the reverse transcrip...

Embodiment 2

[0149] Embodiment 2: Construction of expression vector

[0150] 1. according to the nucleotide sequence of the cow prothymosin gene that embodiment 1 obtains, design primer:

[0151] Forward primer 1': ATG TCA GAC GCG GCC GTG

[0152] Reverse primer 2': CTC GAG CTA GTC ATC TTT ATC AGT CTT C

[0153] 2. Obtaining the Complete Prothymosin Gene

[0154] The polymerase chain reaction was carried out using the recombinant plasmid containing the cow prothymosin gene as a template.

[0155] (1) Reaction system:

[0156] 10x reaction buffer 5 μl

[0157] 25mM MgCl2 4μl

[0158] 10mM dNTP 1μl

[0159] Forward primer 1' 5p mol

[0160] Reverse primer 2' 5p mol

[0161] Plasmid DNA 2μl

[0162] Taq DNA polymerase 0.5μl

[0163] Sterile water 36.5μl

[0164] Total volume 50μl

[0165] (2) PCR reaction conditions: 94° C. for 3 minutes. Cycle: 94°C for 45 seconds, 51°C for 50 seconds, 72°C for 40 seconds, a total of 30 cycles. Extension at 72°C for 5 minutes.

[0166] 3. Liga...

Embodiment 3

[0168] Example 3: Induced protein expression and identification of expression products in eukaryotic yeast cells

[0169] 1. Induce the expression of thymosin in BMGY medium containing 20ml / L methanol, take 1ml of cell culture medium every day, induce expression for 7 days, and save the samples for future use.

[0170] 2. Isolation and Purification of Prothymosin Protein

[0171] The collected cell culture solution was centrifuged at 9000rpm for 20 minutes, and the supernatant was collected and passed through Ni 2+ -The protein was purified by chelatingSepharose affinity column chromatography, and the elution peak was collected for activity determination.

[0172] 3. Determination of molecular weight: SDS-PAGE urea method. After the sample was subjected to SDS polyacrylamide electrophoresis and silver staining, the molecular weight of the expressed product was determined to be 1.3×10 according to the standard protein 4 d.

[0173] 4. Determination of protein content was ca...

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Abstract

The present invention belongs to the field of molecular biology and biological technology. From thymus tissue of milk cow, the milk cow prothoracic gland hormone coding gene is separated. Specific primer pair for inverse transcription and PCR proliferation is designed, and the milk cow prothoracic gland hormone coding gene separated via inverse transcription and PCR has sequence length of 330 bases. The expression vector is further constituted for transforming host cell to obtain prothoracic gland hormone protein with high activity from the host cell culture liquid. The recombinant gene of the present invention may be used in the industrial production of thymosin as one medicine product.

Description

(1) Technical field [0001] The invention relates to isolating prothymosin α gene from cow thymus tissue, its cloning, recombination and expression method and the application of the expression product, and belongs to the field of molecular biology and biotechnology. (2) Background technology [0002] The thymus is the central organ of the body's immune system. The thymosin secreted by it is a group of active peptide substances, which are divided into α and β group thymosins. In the treatment of diseases, α-thymosin is mainly used. Thymosin mainly acts on T cells, promotes the differentiation and maturation of T cells, regulates the production level of B cell antibodies, maintains the balance of the body's immune system, improves the activity of killer cells and macrophages, and promotes the production of interleukin-2 and interferon-α. Synthesis etc. Prothymosin α has multiple and approximately the same effects as thymosin α1, such as the treatment of systemic lupus erythem...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12N15/11C12P19/34
Inventor 张显升魏艳丽李红梅任艳张大伟杨合同
Owner BIOLOGY INST OF SHANDONG ACAD OF SCI
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