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Bactrian camel pepsin A protein gene, recombinant protein and cloning method thereof

A technology of recombinant protein and Bactrian camel, applied in the field of genetic engineering, can solve the problems of high price and limited quantity

Inactive Publication Date: 2013-04-03
XINJIANG WANGYUAN CAMEL MILK IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the enzyme has shown a certain application potential in the clarification of beer, fruit wine, etc. The traditional enzymatic cheese processing uses calf rennet, which is limited in quantity and expensive

Method used

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  • Bactrian camel pepsin A protein gene, recombinant protein and cloning method thereof
  • Bactrian camel pepsin A protein gene, recombinant protein and cloning method thereof
  • Bactrian camel pepsin A protein gene, recombinant protein and cloning method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, a Bactrian camel pepsin A protein gene, the nucleotide sequence of the gene has the sequence shown in SEQ ID NO.1. The new gene sequence obtained by reverse transcription PCR was performed by using the total RNA in the liver tissue of the Bactrian camel as a template and referring to the homologous sequence of the pepsin A protein gene of bovine, human, mouse and other species to design primers. The obtained Bactrian camel pepsin A protein gene cDNA sequence is shown in SEQ ID NO.1.

[0030] According to actual needs, the above-mentioned bactrian camel pepsin A protein gene can be further optimized or / and improved:

[0031] The nucleotide sequence of bactrian camel pepsin A protein gene has the sequence of positions 32-1204 in SEQ ID NO.1. In SEQ ID NO.1, the RT-PCR product length is 1221bp, and the electrophoresis results are shown in the attachment figure 1 , Where 32-34 is the start codon ATG, 1202-1204 is the stop codon TGA, and 32-1204 is the protein coding ...

Embodiment 2

[0032] Example 2. A recombinant protein of pepsin A protein gene of Bactrian camel.

[0033] According to actual needs, the recombinant protein of the above-mentioned bactrian camel pepsin A protein gene can be further optimized or / and improved:

[0034] The amino acid sequence of the recombinant protein has the sequence shown in SEQ ID NO.2. The coding sequence (CDS) in the Bactrian camel pepsin A protein gene cDNA sequence is translated into the protein coding sequence according to the universal codon, see SEQ ID NO.2.

[0035] The recombinant protein is a polypeptide having the amino acid sequence shown in SEQ ID NO. 2, or a conservative variant polypeptide, or an active fragment, or an active derivative thereof.

[0036] The Bactrian camel myosin gene was obtained from the Bactrian camel pepsin A protein by designing a pair of primers. The nucleotide sequence information of the primer pair is as follows:

Embodiment 3

[0037] Example 3, a method for cloning the pepsin A protein gene of Bactrian camel, characterized by the following steps:

[0038] The first step, total RNA extraction

[0039] 1. Isolation

[0040] Bactrian camels were collected from Alxa League in Inner Mongolia, slaughtered quickly, and liver samples were taken. The tissue samples were quickly frozen in liquid nitrogen and stored at -70°C, and they were taken back to the laboratory to prepare for the extraction of total tissue RNA.

[0041] 2. Total RNA isolation

[0042] (1) Preparation for RNA extraction

[0043] The glassware was soaked in 0.1M NaOH overnight, rinsed repeatedly with tap water, rinsed twice with distilled water, and baked at 180°C for 4 hours.

[0044] The homogenizer and electrophoresis tank are soaked in 3% hydrogen peroxide for 20-30 minutes, and then rinsed with 0.1% DEPC water. Since the uninactivated DEPC has an effect on the PCR reaction, it can be treated with 0.5% SDS.

[0045] Tip and EP tubes were soaked i...

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Abstract

The invention relates to the technical field of genetic engineering and discloses a bactrian camel pepsin A protein gene, a recombinant protein and a cloning method thereof. The bactrian camel pepsin A protein is a protein with multiple biological functions and has a key regulation function of regulating protein digestion; and therefore, due to the research of pepsin A from genes, proteins, enzymatic activity, inhibitors and the like, a novel enlightenment is brought to development of the fields of medicines and foods. The pepsin A gene sequence of other species is referred, a cDNA sequence of the bactrian camel pepsin A gene is obtained through clone sequencing, the CDS sequence of the pepsin A gene of various species is subjected to homologous comparison, the structural features of the pepsin A gene are known and verified, and basic data are provided for a relationship for researching the pepsin A gene and the bactrian camel digestion system.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and relates to a Bactrian camel pepsin A protein gene, a recombinant protein and a cloning method thereof. Background technique [0002] Pepsin (English name: Pepsin) is a digestive protease secreted by the gastric chief cell in the stomach. Its function is to decompose the protein in food into small peptide fragments. The precursor of pepsin is called pepsinogen. Pepsin has different pepsin components in different biological species, and its molecular weight and amino acid composition are different. There are certain differences, but they also have great similarities. [0003] In vertebrates, pepsin belongs to the aspartic protease family, which includes cathepsin, renin (angiotensinogenase), etc. in addition to pepsin, so the aspartic protease family is a protein superfamily. Vincenzo Carginale et al. studied the relationship between the selective evolution and functional diversit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/64C12N15/10
Inventor 陈钢粮
Owner XINJIANG WANGYUAN CAMEL MILK IND
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