阅读说明：本技术 一种降低乳糖含量的牛奶制作方法 (Milk production method capable of reducing lactose content ) 是由 刘羿羿 吴耀 张东 荆文魁 张立 苏小虎 张焱如 李璐 王申元 周欢敏 曹俊伟 于 2018-08-23 设计创作，主要内容包括：本发明公开了一种降低乳糖含量的牛奶制作方法,属于基因工程技术领域。本发明利用了耐热的硫矿硫化叶菌的嗜热菌糖苷酶基因(LacS)可以耐受80℃以上高温的特性,通过乳腺生物反应器生产外源蛋白的技术将该基因序列进行密码子优化后的lacs基因转染到牛的乳腺细胞进行特异性的表达,使生产出的牛乳含有乳糖分解酶,经常规加热消毒后牛乳中的乳糖被降解,含量下降。此方法具有成本低、产量稳定、安全高效等优点,对于工业制备低乳糖牛奶具有颠覆性的意义。(The invention discloses a milk preparation method for reducing lactose content, and belongs to the technical field of genetic engineering. The invention utilizes the characteristic that thermophilic mycose glycosidase gene (LacS) of heat-resistant sulfolobus solfataricus can resist high temperature of more than 80 ℃, and the LacS gene of which the gene sequence is subjected to codon optimization is transfected into mammary gland cells of a cow to perform specific expression by a technology for producing exogenous protein by a mammary gland bioreactor, so that the produced cow milk contains lactose clastic enzyme, and lactose in the cow milk is degraded and reduced after conventional heating and sterilization. The method has the advantages of low cost, stable yield, safety, high efficiency and the like, and has subversive significance for industrially preparing the low-lactose milk.)

1. A milk preparation method for reducing lactose content is characterized by comprising the following steps:

(1) constructing a specific expression vector of an exogenous gene, wherein the exogenous gene is a gene for coding glycosidase;

(2) transfection: transferring the specific expression vector containing the exogenous gene into a fibroblast line derived from the skin tissue of the milk cow fetus;

(3) screening a cell line with successful transfection, and carrying out expanded culture;

(4) cloning the cell line obtained by screening in the step (3) to obtain a milk cow carrying the exogenous gene, and feeding the milk cow to enable the milk cow to produce milk;

(5) heating the milk.

2. The method according to claim 1, wherein the foreign gene is LacS gene obtained by codon optimization of a thermophilic glycosylase gene (LacS) sequence from sulfolobus solfataricus, and the nucleotide sequence of the LacS gene is shown as SEQ ID No. 1.

3. The method of claim 1 or 2, wherein the exogenous specific expression vector is a vector pMD19-T expressing lacs gene encoding glycosidase.

4. The method as claimed in claim 1, wherein the transfection in step (2) is performed at a voltage of 175-200V.

5. The method according to claim 1, wherein step (5) is heating the milk at 75-100 ℃.

6. A dairy product produced according to the method of claim 1.

7. A recombinant gene expression vector is characterized in that pMD19-T is used as a vector to express lacs gene for coding glycosidase, and the nucleotide sequence of the lacs gene is shown as SEQ ID NO. 1.

8. Use of the recombinant gene expression vector of claim 7 in the preparation of a dairy product.

Technical Field

The invention relates to a milk preparation method for reducing lactose content, and belongs to the technical field of genetic engineering.

Background

Lactose intolerance is non-infectious diarrhea caused by the fact that lactase is less secreted and cannot completely digest and decompose lactose in breast milk or cow milk, which is also called lactase deficiency. Lactase deficiency is a worldwide problem, the incidence of the far east population is high, most of the population does not have symptoms, but symptoms such as diarrhea and the like often occur in newborns and infants who mainly drink milk.

According to the findings published by the Food Intololance Network, 95% of Asians suffer from lactose Intolerance. The lactose content in ordinary milk is 4.2%, and lactose intolerance people can not degrade and absorb lactose due to lack of lactose decomposing enzyme. The undegradable lactose is fermented by intestinal microorganisms as carbon compounds to produce acid and gas, resulting in gastrointestinal disturbances and loss of valuable proteins and minerals. In addition, lactose malabsorption also affects the absorption of mineral elements such as iron, zinc, calcium and the like in cow milk.

At present, methods for reducing the lactose content in milk mainly comprise fermenting into yoghourt, sieving or hydrolyzing lactose by adding lactose hydrolase after carrying out bus sterilization on raw milk. The fermentation method changes the flavor of the milk, the milk obtained by the sieving method is mainly made into milk powder, the sale price is higher in the market, and the method of adding the enzyme after the pasteurization has the problems of high energy consumption, low production efficiency and the like.

The animal mammary gland bioreactor is to utilize mammary gland specificity regulating and controlling element to guide the specific expression of exogenous gene in mammary gland and produce recombinant protein with mammary gland tissue of transgenic animal. The basic principle is that DNA recombination technology and transgenic technology are applied to transfer target genes into fertilized eggs in prokaryotic stage or animal embryos in 1-2 cell stage, transgenic mammary gland expression individuals are obtained through embryo transplantation, and target gene expression products are obtained through collecting milk. The animal mammary gland bioreactor is the most studied and widely applied animal bioreactor at present, and is concerned by scientists in various countries.

The exogenous protein produced by the mammary gland bioreactor has wide variety and obvious advantages. Firstly, the animal mammary gland has a complete protein posttranslational modification system comprising glycosylation, phosphorylation, carboxylation and the like, thereby ensuring the high biological activity of the product; secondly, the product of the animal mammary gland bioreactor is directly secreted out of the body by milk, the casein precipitate whey is removed by a conventional method, and the recombinant protein can be obtained by chromatography.

The method for producing protein in large quantity by using the mammary gland bioreactor is widely applied, but the transfection efficiency is reduced due to the larger carrier, and the efficiency of gene integration to genome is lower, so the research of producing lactase by using the cow mammary gland bioreactor does not appear at present.

Disclosure of Invention

The invention aims to provide a method for preparing milk with reduced lactose content, which is to transfect an exogenous gene into mammary cells of a cow for specific expression so that the produced milk contains lactase, and specifically comprises the following steps:

(2) transfection: transferring the specific expression vector containing the exogenous gene into a cell line derived from the skin tissue of the milk cow fetus;

(3) screening a cell line with successful transfection, and carrying out expanded culture;

(4) producing milk cows carrying exogenous genes by a cloning method to produce milk;

(5) heating milk at 75-100 deg.C.

The exogenous gene is a LacS gene obtained by performing codon optimization on a thermophilic bacterial glycosidase gene (LacS) sequence from sulfolobus solfataricus (P2), and the nucleotide sequence of the exogenous gene is shown as SEQ ID No. 1. The lacs gene is introduced into an animal body, and the specific expression part is in a mammary gland.

In one embodiment of the invention, the specific expression vector expresses lacs gene by using a pMD19-T vector framework, and the specific expression vector further comprises a β -casein promoter, TALEN enzyme digestion recognition sites and a micro homologous site thereof.

In one embodiment of the present invention, the cell line in step (2) is a fibroblast cell line which is derived from skin tissue of a cow fetus, can be continuously passaged for 15 generations, has normal cell cycle and karyotype analysis, and has good activity.

In one embodiment of the present invention, the transfection voltage is set to 175-200V for the transfection in step (2).

In one embodiment of the invention, step (5) is heating the milk at 75-100 ℃.

The invention also aims to provide a gene expression vector which takes a pMD19-T vector as a framework and takes a target gene as a lacs gene with a nucleotide sequence shown in SEQ ID NO.1 and application thereof in the preparation of dairy products.

The invention utilizes the characteristic that thermophilic mycose glycosidase gene (LacS) of heat-resistant sulfolobus solfataricus can resist high temperature of more than 80 ℃, and the LacS gene of which the gene sequence is subjected to codon optimization is transfected into mammary gland cells of a cow to perform specific expression by a technology for producing exogenous protein by a mammary gland bioreactor, so that the produced cow milk contains lactose clastic enzyme, and lactose in the cow milk is degraded and reduced after conventional heating and sterilization. The method has the advantages of low cost, stable yield, safety, high efficiency and the like, and has subversive significance for industrially preparing the low-lactose milk.

Drawings

FIG. 1: TALEN plasmid map (carrying CMV promoter, Sp6 in vitro transcription promoter);

FIG. 2: lacs gene specific expression vector frameworks;

FIG. 3: identifying an electrophoretogram of the PCR of the product 1-5; the template is a cell after expanded culture, and the target product is a partial fragment of lacs gene; identifying primers P3, P4; the length of the target fragment is about 560 bp; m is 100bp ladder marker;

FIG. 4: PCR identification electrophoretogram of the product 6-11; the template is a cell after expanded culture, and the target product is lacs gene and a downstream part sequence of a cell genome insertion site; identifying primers P3, P2; the length of the target fragment is about 900 bp; m is 100bpladder marker.

Detailed Description