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CN-122012566-A - Recombinant vector and recombinant strain for efficiently expressing lipase, and construction method and application thereof

CN122012566ACN 122012566 ACN122012566 ACN 122012566ACN-122012566-A

Abstract

The invention discloses a recombinant vector for efficiently expressing lipase, a recombinant strain, a construction method and application thereof, and relates to the technical field of molecular biology. The BSFP-0720 promoter sequence in the recombinant vector can promote cells containing the recombinant vector to overexpress lipase coded by the lipA gene under the condition of no addition of an inducer, and in addition, the co-expression of the lipA gene sequence and the lipH gene sequence promotes the correct folding of the lipase coded by the lipA gene sequence after the expression and the maturation and secretion of the lipase, thereby improving the expression quantity of the lipase. After the recombinant vector is expressed in COO gene knockout strain, the purification process of lipase is shortened, the purity of lipase is improved, and the lipase is applied to an in vitro detection kit, so that the detection accuracy is high.

Inventors

  • LIU YUNBO
  • ZHU PAN
  • SHI YONG
  • XUE JUN
  • Mi Qianfen
  • WU HONGMEI
  • JI CHENGDONG
  • ZHAO ZHONGHAO
  • YI WEIJING

Assignees

  • 中元汇吉生物技术股份有限公司

Dates

Publication Date
20260512
Application Date
20241111

Claims (10)

  1. 1. A recombinant vector for efficiently expressing lipase is characterized by comprising a nucleotide sequence encoding lipA, a nucleotide sequence encoding lipH and a BSFP _0720 promoter.
  2. 2. The recombinant vector of claim 1, wherein the lipA and/or lipH gene is derived from pseudomonas aeruginosa; preferably, the nucleotide sequence of the BSFP _0720 promoter is shown as SEQ ID NO. 1; and/or, the nucleotide sequence for coding lipA is shown as SEQ ID NO. 2; and/or the nucleotide sequence of the coding lipH is shown as SEQ ID NO. 3.
  3. 3. The recombinant vector according to claim 1 or 2, wherein the recombinant vector is an integrative plasmid vector pBBR1MCS-2, pUCP vector or pTAC vector; preferably, the recombinant vector is an integrative plasmid vector pBBR1MCS-2.
  4. 4. A recombinant strain that expresses a lipase at high efficiency, characterized in that the recombinant strain comprises the recombinant vector of any one of claims 1 to 3; Preferably, the strain source of the recombinant strain is pseudomonas aeruginosa.
  5. 5. The recombinant strain of claim 4, wherein the recombinant strain is incapable of expressing cholesterol oxidase; preferably, the strain is Pseudomonas aeruginosa with cholesterol oxidase gene knocked out.
  6. 6. A method of constructing a recombinant strain, comprising: (1) Constructing a recombinant vector according to any one of claims 1 to 3; (2) Transforming the recombinant vector into pseudomonas aeruginosa; Preferably, the pseudomonas aeruginosa is incapable of expressing cholesterol oxidase; More preferably, the pseudomonas aeruginosa is a strain that knocks out cholesterol oxidase genes.
  7. 7. A method for efficient expression of lipase, comprising: (1) Culturing the recombinant strain of claim 5 under conditions conducive to lipase expression; (2) And separating and purifying lipase by utilizing the cultured recombinant strain.
  8. 8. Use of a recombinant vector according to any one of claims 1 to 3, or a recombinant strain according to claim 4 or 5, for the preparation of a lipase.
  9. 9. A lipase, which is characterized in that, the lipase prepared by the method of claim 7.
  10. 10. Use of the lipase of claim 9 in the preparation of an in vitro diagnostic reagent; preferably, the in vitro diagnostic reagent comprises a triglyceride detection reagent or a cholesterol detection reagent; More preferably, the in vitro diagnostic reagents include total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol and small dense low density lipoprotein cholesterol detection reagents.

Description

Recombinant vector and recombinant strain for efficiently expressing lipase, and construction method and application thereof Technical Field The invention relates to the technical field of molecular biology, in particular to a recombinant vector and recombinant strain for efficiently expressing lipase, and a construction method and application thereof. Background Lipases (EC 3.1.1.3) are a class of hydrolases which catalyze the decomposition of triglycerides and are widely distributed in higher organisms and microorganisms. In the field of in vitro diagnostics, lipases play a vital role in Triglyceride (TG) detection kits, i.e. during the initial reaction, they hydrolyze triglycerides in serum samples into glycerol and fatty acids, providing reaction substrates for subsequent reactions. Therefore, lipases are of great commercial value as core materials for diagnostic reagents due to their efficient, high quality, low cost industrial production. Currently, the method for obtaining lipase mainly comprises (1) directly and naturally extracting lipase from microorganism, and (2) inducing microorganism to express lipase by using substrate such as olive oil. However, both methods have the disadvantages of low yield and high cost. Disclosure of Invention The invention mainly aims to provide a recombinant vector and recombinant strain for efficiently expressing lipase, and a construction method and application thereof, and aims to solve the problems of low yield and high cost of a method for obtaining lipase in the prior art. In order to achieve the aim, the invention provides a recombinant vector for efficiently expressing lipase, which comprises a nucleotide sequence for coding lipA, a nucleotide sequence for coding lipH and a BSFP _0720 promoter. In one embodiment, the lipA and/or lipH gene is derived from pseudomonas aeruginosa; preferably, the nucleotide sequence of the BSFP _0720 promoter is shown as SEQ ID NO. 1; and/or, the nucleotide sequence for coding lipA is shown as SEQ ID NO. 2; and/or the nucleotide sequence of the coding lipH is shown as SEQ ID NO. 3. In one embodiment, the recombinant vector is an integrative plasmid vector pBBR1MCS-2, pUCP vector or pTAC vector; preferably, the recombinant vector is an integrative plasmid vector pBBR1MCS-2. The invention also provides a recombinant strain for efficiently expressing lipase, wherein the recombinant strain comprises the recombinant vector; preferably, the recombinant strain is derived from Pseudomonas aeruginosa. In one embodiment, the recombinant strain is incapable of expressing cholesterol oxidase; preferably, the strain is Pseudomonas aeruginosa with cholesterol oxidase gene knocked out. The invention also provides a construction method of the recombinant strain, which comprises the following steps: (1) Constructing the recombinant vector; (2) Transforming the recombinant vector into pseudomonas aeruginosa; Preferably, the pseudomonas aeruginosa is incapable of expressing cholesterol oxidase; More preferably, the pseudomonas aeruginosa is a strain that knocks out cholesterol oxidase genes. The invention also provides a method for efficiently expressing lipase, which comprises the following steps: (1) Culturing the recombinant strain under conditions conducive to lipase expression; (2) And separating and purifying lipase by utilizing the cultured recombinant strain. The invention also provides an application of the recombinant vector or the recombinant strain in preparing lipase. The invention also provides lipase, which is prepared by the method. The invention also provides application of the lipase in preparing an in-vitro diagnostic reagent; preferably, the in vitro diagnostic reagent comprises a triglyceride detection reagent or a cholesterol detection reagent; More preferably, the in vitro diagnostic reagents include total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol and small dense low density lipoprotein cholesterol detection reagents. In the technical scheme of the invention, the BSFP _0720 promoter sequence in the recombinant vector can promote cells containing the recombinant vector to overexpress lipase coded by the lipA gene under the condition of no addition of an inducer, and in addition, the lipA gene sequence and the lipH gene sequence are coexpressed, so that correct folding of the lipase coded by the lipA gene sequence after expression is promoted, maturation and secretion of the lipase are promoted, and the expression quantity of the lipase is improved. After the recombinant vector is expressed in a cholesterol oxidase (COO) gene knockout strain, COO removal steps in the lipase purification process can be avoided, the purification treatment steps are simplified, and the purity of the lipase is improved, so that the lipase disclosed by the invention is suitable for being applied to an in-vitro detection kit, and the detection accuracy is high. Drawings In order to more clearly illustrate