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Recombinant chymosin of abomasum of yak and preparation method thereof

A technology of rennet and recombinant bacteria, which is applied in the field of genetic engineering, can solve the problems of affecting cheese production, cheese bitterness, high cost of yak abomasum rennet, and achieve good market application prospects and high enzyme activity.

Inactive Publication Date: 2015-02-04
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of rennet contains proteases of low specificity and may cause a bitter taste or affect cheese yield
[0004] Yak is a precious and rare herbivorous domestic animal in the Qinghai-Tibet Plateau. The cost of yak abomasum rennet extracted directly from yak abomasum is high. The preparation of yak abomasum rennet by genetic engineering can reduce the cost. However, the preparation of A recombinant bacterium that can express active yak abomasum chymosin is not easy, and there is no report on the preparation of yak abomasum chymosin by genetic engineering

Method used

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  • Recombinant chymosin of abomasum of yak and preparation method thereof
  • Recombinant chymosin of abomasum of yak and preparation method thereof
  • Recombinant chymosin of abomasum of yak and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 Preparation of recombinant protein of the present invention

[0031] 1. Recombinant expression

[0032] (1) Cloning of prochymosin gene

[0033] The abomasum tissue was collected from the abomasum of a newly slaughtered lactating calf. After washing with DEPC water, the abomasum tissue was immediately put into liquid nitrogen for preservation. Total RNA was extracted according to the instructions of Trizol reagent (Invitrogen, USA). The concentration and quality of total RNA were analyzed by UV spectrophotometer. For first-strand cDNA synthesis RT-PCR kit (TAKARA, Dalian, China), and synthesized according to the manufacturer's instructions.

[0034] The prorennin gene sequence was amplified from the pMD19-T-Pro plasmid by the designed primers, and the primer sequences are shown in Table 1 below:

[0035] Table 1 Primer sequences used in the study

[0036]

[0037] The reaction system is as follows: 1 μl of cDNA template, 2.5 μl of 10×Ex Taq Buffer...

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Abstract

The invention discloses a nucleotide sequence shown in SEQ ID NO: 1, a recombinant vector and a recombinant bacterium comprising the nucleotide sequence as well as chymosin which is encoded by the nucleotide sequence shown in SEQ ID NO: 1. The invention further discloses a method for preparing recombinant chymosin of abomasum of a yak by using the recombinant bacterium. The recombinant chymosin of abomasum of the yak disclosed by the invention is soluble and high in activity and has an industrial application value.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant yak abomasum rennet and a preparation method thereof. Background technique [0002] Rennet belongs to acid protease, which is an aspartic acid protease originally found in calf abomasum, which can act on Phe in k-casein molecule 105 -Met 106 peptide bonds, forming insoluble k-casein, which eventually causes milk to clot. Therefore, it is widely used in the pretreatment steps of different kinds of traditional cheese making. Chymosin is synthesized as a precursor protein (prochymosin precursor, 381 amino acids) containing a 16 amino acid signal peptide. After cleavage of this signal peptide, prochymosin is formed, and then under the acidic conditions in the stomach, the protein forms active chymosin after 42 amino acids are cleaved from the N-terminus. [0003] There are four sources of commercially used rennet: animals, plants, microorganisms and recombinant re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N15/81C12N1/19C12N9/64C12R1/84
Inventor 江明锋罗樊
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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