阅读说明：本技术 一种适用于枯草芽孢杆菌分泌表达蛋白的表达载体及应用 (Expression vector suitable for bacillus subtilis secretion expression protein and application ) 是由 饶志明 李谞 张显 朱曼迟 杨套伟 徐美娟 邵明龙 杜宇轩 贾以泽 王嘉轩 于 2019-10-29 设计创作，主要内容包括：本发明公开了一种适用于枯草芽孢杆菌分泌表达蛋白的表达载体及应用,属于基因工程和生物技术领域。该分泌载体是将编码信号肽SP<Sub>phoD</Sub>的基因(核苷酸序列如SEQ ID NO.1)和编码分子伴侣PrsA的基因(核苷酸序列如SEQ ID NO.3)分别连接到pMA5-P43上构建分泌质粒pMA5-SPP。以pMA5-SPP为载体,在枯草芽孢杆菌中表达L-天冬酰胺酶,其胞外分泌量占总表达量的38％,胞外分泌量是常见信号肽SP<Sub>pel</Sub>介导下分泌量的2.95倍。(The invention discloses an expression vector suitable for bacillus subtilis to secrete expression protein and application thereof, belonging to the field of genetic engineering and biotechnology. The secretion carrier is a polypeptide which encodes a signal peptide SP phoD The gene (the nucleotide sequence is shown as SEQ ID NO.1) and the gene (the nucleotide sequence is shown as SEQ ID NO.3) for coding the molecular chaperone PrsA are respectively connected to pMA5-P43 to construct a secretion plasmid pMA 5-SPP. pMA5-SPP is used as a vector to express L-asparaginase in bacillus subtilis, the extracellular secretion amount accounts for 38 percent of the total expression amount, and the extracellular secretion amount is common signal peptide SP pel 2.95 times of the amount secreted under mediation.)

1. An expression vector is characterized in that a gene coding a signal peptide SPphoD and a gene coding a molecular chaperone PrsA are connected to a vector pMA5-P43 to obtain an expression vector pMA 5-SPP; the amino acid sequence of the signal peptide SPphoD is shown as SEQ ID NO.1, and the amino acid sequence of the molecular chaperone PrsA is shown as SEQ ID NO. 3.

2. The expression vector of claim 1, wherein the gene encoding the signal peptide SPphoD is ligated into the vector pMA5-P43 between the multiple cloning sites EcoR V and Kpn I, and expressed under the mediation of the P43 promoter.

3. The expression vector of claim 1, wherein the gene encoding chaperone PrsA is ligated into the vector pMA5-P43 between the multiple cloning sites BamH I and Mlu I, and expressed under the mediation of the PpHpaII promoter.

4. The expression vector of any one of claims 1 to 3, wherein the nucleotide sequence of the gene encoding the signal peptide SPphoD is shown in SEQ ID No.2, and the nucleotide sequence of the gene encoding the chaperone PrsA is shown in SEQ ID No. 4.

5. A genetically engineered bacterium characterized in that the expression vector according to any one of claims 1 to 4 is used as an expression vector.

6. The genetically engineered bacterium of claim 5, wherein the genetically engineered bacterium is hosted in Bacillus subtilis 168.

7. The genetically engineered bacterium of claim 5, wherein the genetically engineered bacterium expresses an L-asparaginase whose amino acid sequence is shown as SEQ ID No. 13.

8. The genetically engineered bacterium of claim 6, which is prepared by ligating a gene encoding L-asparaginase between restriction sites Kpn I and Hind III of pMA5-SPP to give a recombinant plasmid, and transforming the recombinant plasmid into Bacillus subtilis 168.

9. A method for preparing L-asparaginase, which is characterized in that the genetic engineering bacteria of claim 7 or 8 are inoculated in an LB culture medium and cultured for 8-12h at 35-39 ℃ and 220rpm which is 200-; inoculating to new LB culture medium in an amount of 0.5-5%, and culturing at 35-39 deg.C for 20-30 h.

10. Use of the expression vector of claims 1-4 for the production of an endogenous or exogenous protein.

Technical Field

The invention relates to an expression vector suitable for bacillus subtilis to secrete expression protein and application thereof, belonging to the field of genetic engineering and biotechnology.

Background

In the process of expressing recombinant protein by using microorganisms, good secretion performance reduces the recovery cost of products, and is an important consideration factor for whether the recombinant protein can be industrially produced. The signal peptide is a peptide chain at the N end of the secretory protein and can mediate the protein to be secreted to the outside of cells, and has important significance on the secretory expression of the protein. Molecular chaperones are capable of recognizing and binding to incompletely folded or assembled proteins in cells, and help these proteins to fold and secrete (Molecular microbiology,2010,8(4): 727-37; Microbial cell factors, 2015,14, 92). Therefore, in constructing a secretion expression vector of the protein, selecting a proper signal peptide and a proper molecular chaperone has important significance.

Bacillus subtilis has good secretion capacity due to lack of cell outer membrane, and is an ideal host for secreting biological products. Meanwhile, the Bacillus subtilis also has the characteristics of food safety, clear and good production technology and fermentation foundation of genetic information and the like, so the Bacillus subtilis is widely applied to the biological fermentation preparation of proteins, food additives and antibiotics (Trends in biotechnology,1992,10(7): 247-56; Journal of clinical microbiology,1998,36(1): 325-6; Nature,1997,390(6657): 249-56).

In the process of secreting and expressing foreign protein by taking bacillus subtilis as host bacteria, the construction of a carrier containing effective signal peptide and molecular chaperone has important significance for protein secretion. At present, no signal peptide SP is constructed on a carrier at the same time_phoDGene and molecular chaperone PrsA gene for increasing bacillus subtilisLiterature reports on the secretion levels of bacillus proteins.

There are relatively few reports on extracellular secretion of L-asparaginase, And studies show that Bacillus subtilis B11-06L-asparaginase is expressed by Bacillus subtilis 168 as a host bacterium, And that extracellular enzyme activity accounts for 57.1% of total enzyme activity (journal of Agricultural And Food Chemistry,2013,61(39):9428-9434), but recombinase enzyme activity is only 9.98U/mL. Bacillus subtilis 168 is used as a host bacterium to express Pyrococcus yayanosii-derived L-asparaginase, the extracellular and intracellular activities of which are respectively 23.31U/mL and 65.72U/mL (Scientific Reports,2018,8(1):7915), and the enzyme activity level is limited.

Therefore, the method for further improving the extracellular secretion level of the L-asparaginase has important application value for industrial preparation of the L-asparaginase.

Disclosure of Invention

The first purpose of the invention is to provide a bacillus subtilis expression vector, which is obtained by connecting a gene coding a signal peptide SPphoD and a gene coding a molecular chaperone PrsA to a vector pMA5-P43 to obtain an expression vector pMA 5-SPP; the amino acid sequence of the signal peptide SPphoD is shown as SEQ ID NO.1, and the amino acid sequence of the molecular chaperone PrsA is shown as SEQ ID NO. 3. In one embodiment of the invention, the gene encoding the signal peptide SPphoD is ligated into the vector pMA5-P43 between the multiple cloning sites EcoR V and Kpn I, expressed under the mediation of the P43 promoter, and the gene encoding the chaperone PrsA is ligated into the vector pMA5-P43 between the multiple cloning sites BamH I and Mlu I, expressed under the mediation of the PpHpaII promoter.

The pMA5-P43 plasmid is obtained by connecting a promoter P43 to a pMA5 vector by using enzyme digestion connection.

The construction method of the pMA5-P43 plasmid is characterized in that a forward primer F (nucleotide sequence is shown as SEQ ID NO. 15) and a reverse primer R (nucleotide sequence is shown as SEQ ID NO. 16) are used for amplifying a promoter P43 from a bacillus subtilis 168 genome; the vector pMA5 was double digested with restriction enzymes EcoR I and Hind III; using a homologous recombination kit (MultiS One-Step Cloning Kit, Novozam) ligated promoter P43 between the multiple Cloning sites EcoR I and Hind III of pMA5 to give recombinant plasmid pMA 5-P43.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the signal peptide SPphoD is shown in SEQ ID NO. 2.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the molecular chaperone PrsA is shown in SEQ ID NO. 4.

The second purpose of the invention is to provide a genetically engineered bacterium, which takes the expression vector as an expression vector.

In one embodiment of the present invention, the genetically engineered bacterium is a bacillus subtilis host.

In one embodiment of the invention, the genetically engineered bacteria express an L-asparaginase whose amino acid sequence is shown in SEQ ID No. 13.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the L-asparaginase is shown as SEQ ID No. 14.

The third purpose of the invention is to provide the preparation method of the gene engineering bacteria, which is to connect the gene coding the L-asparaginase between the restriction sites Kpn I and Hind III of pMA5-SPP to obtain a recombinant plasmid, and transform the recombinant plasmid into the bacillus subtilis 168 to obtain the bacillus subtilis gene engineering bacteria.

The fourth purpose of the invention is to provide a method for preparing L-asparaginase, which comprises the steps of inoculating the genetic engineering bacteria into an LB culture medium, culturing at 35-39 ℃, 200-rpm and 220-12 h, transferring into a new LB culture medium according to 0.5-5% of the inoculum size, culturing at 35-39 ℃ for 20-30h, centrifuging the fermentation liquor, taking the supernatant as extracellular crude enzyme liquid, and taking the cell disruption supernatant as intracellular crude enzyme liquid.

The fifth purpose of the invention is to provide the application of the bacillus subtilis expression vector in preparing endogenous protein or exogenous protein.

The signal peptide SPphoD is used for mediating protein secretion, the molecular chaperone PrsA is co-expressed for assisting protein to be transported out of a cell membrane, the extracellular secretion amount of the L-asparaginase is 38% of the total expression amount, and compared with the secretion expression mediated by the common signal peptide SPpel, the extracellular secretion amount of the L-asparaginase is improved by 2.95 times by the method provided by the invention.

Detailed Description