CN116144690A - A shuttle vector pDXA between Escherichia coli and lactic acid bacteria and its construction method and application - Google Patents

Abstract

The invention discloses a shuttle vector pDXA of escherichia coli and lactobacillus, a construction method and application thereof, and belongs to the field of molecular biology. The shuttle vector pDXA is constructed and obtained by synthesizing ampicillin resistance gene and promoter and pMB1 replicon sequence through genes and connecting the genes to lactobacillus acidophilus plasmid pDX. The shuttle vector pDXA of the present invention can replicate and express foreign genes in E.coli and various lactic acid bacteria. The plasmid vector is used for completing the cloning of target genes and the construction of vectors in an escherichia coli host, thereby facilitating the development of functional gene expression research and lactobacillus genetic modification in lactobacillus.

Description

A shuttle vector pDXA between Escherichia coli and lactic acid bacteria and its construction method and application

technical field

The invention belongs to the field of molecular biology, and in particular relates to a shuttle vector pDXA between Escherichia coli and lactic acid bacteria, its construction method and application.

Background technique

Lactic acid bacteria is a general term for various bacteria that can use carbohydrate fermentation to produce lactic acid. It exists widely in nature and is distributed in soil, lakes, animals, plants and fermented foods. Lactic acid bacteria are a kind of probiotics. Lactic acid bacteria in the human digestive system can adjust the micro-ecological balance of the flora, promote the digestion and absorption of nutrients such as lactose, inhibit the growth of spoilage bacteria, eliminate the toxins produced by spoilage bacteria, and improve the function of the gastrointestinal tract. Enhance the body's immunity, etc. In addition, lactic acid bacteria have high application value in fields such as agriculture, food and medicine. Lactobacillus acidophilus belongs to the genus Lactobacillus and is widely used in various functional foods. It is a food-grade probiotic that attaches great importance to research and development among lactic acid bacteria.

Plasmids are closed circular DNA molecules with autonomous replication ability that exist in the cytoplasm outside the chromosomes of microorganisms such as bacteria, yeasts, and actinomycetes. Most of the coding features of the plasmid are beneficial to the strain and can improve the survival advantage of the strain. Lactic acid bacteria can carry multiple plasmids of different sizes, most of which are cryptic plasmids, and some other plasmids have certain special functions. Plasmids are often used to construct vectors for the shuttle between strains. Gene cloning in Escherichia coli is more mature and convenient, the transformation of lactic acid bacteria is more complicated than that of Escherichia coli, and the transformation efficiency is relatively low, constructing a vector that can shuttle between Escherichia coli and lactic acid bacteria can improve the genetic engineering operation in lactic acid bacteria s efficiency.

Contents of the invention

The object of the present invention is to provide a carrier that shuttles between Escherichia coli and lactic acid bacteria, so as to solve the difficult problem of heterologous gene introduction in lactic acid bacteria. Another object of the present invention is to provide a lactic acid bacteria promoter for E. coli expression.

The present invention provides a vector for shuttling between Escherichia coli and lactic acid bacteria, the shuttle vector is obtained by linking the ampicillin resistance gene and promoter and pMB1 replicon sequence into the Lactobacillus acidophilus plasmid pDX, the shuttle vector pDXA base The sequence is shown in SEQ ID NO:1; the sequence of the plasmid pDX is shown in SEQ ID NO:2.

The invention provides the application of the shuttle vector, and the shuttle vector pDXA of the invention can express the genes of phytase, protease, amylase and cellulase in Escherichia coli.

Compared with the prior art, the shuttle vector pDXA of the present invention can replicate in Escherichia coli, Lactococcus lactis, Lactococcus lactis subspecies, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei And express foreign genes, solve the problem of narrow host range of existing plasmid vectors. The shuttle carrier of the present invention can not only shuttle in Escherichia coli and different lactic acid bacteria, but also in Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei Replication inherited to 60 generations. The shuttle vector of the present invention is used to express the enzyme gene in Escherichia coli, and the expression level of the enzyme gene is more than 347% higher than that of the Escherichia coli phosphate glucose isomerase gene promoter. The plasmid vector is used to complete the cloning of the target gene and the construction of the vector in the Escherichia coli host, which is convenient for carrying out functional gene expression research and genetic transformation of the lactic acid bacteria in the lactic acid bacteria.

Description of drawings

Figure 1 shows the plasmid detected by agarose gel electrophoresis; the leftmost lane is the DNA Marker, and the rightmost lane is the extracted plasmid, showing three plasmid bands, and the plasmid pDX is the smallest plasmid band.

Figure 2 is the plasmid pDX map; in the map, dso is the double-strand origin of plasmid replication, sso is the single-strand origin of plasmid replication, and ORF1 rep encodes the replication initiation protein.

Figure 3 is the map of the shuttle vector pDXA; in the map, pMB1 is the replication initiation sequence, and AmpR is the ampicillin resistance gene.

Fig. 4 shows the enzyme gene expressed by the lactic acid bacteria promoter.

Detailed ways

A shuttle vector pDXA between Escherichia coli and lactic acid bacteria of the present invention and its construction method and application will be further described in detail below in conjunction with specific examples.

Embodiment 1: the extraction of plasmid pDX

Culture Lactobacillus acidophilus statically in MRS liquid medium at 37°C for 24 hours, collect the bacteria by centrifugation, and use 50mg/mL lysozyme solution (10mmol/L Tris-HCl, 5mmol/L EDTA, pH 8.0) at 37 Treat at ℃ for 1 h, and extract the plasmid with a plasmid mini-extraction kit. Utilize agarose gel electrophoresis to detect and confirm the size of the plasmid, and the agarose gel electrophoresis result is as shown in Figure 1, and the result shows that there are 3 bands in the plasmid extract, indicating that the bacterial strain carries multiple plasmids, and about 2kb of them in size The small plasmid was purified and recovered, and the plasmid was pDX, and its sequence was determined.

Embodiment 2: the construction of shuttle carrier

By genetically synthesizing the ampicillin resistance gene, promoter and pMB1 replicon sequence, and connecting it to the Lactobacillus acidophilus plasmid pDX, the shuttle vector pDXA is constructed, the plasmid pDX is shown in Figure 2, and the plasmid pDXA is shown in Figure 2 3. A tetracycline resistance gene connected with the promoter of ORF3 in the plasmid pDX was synthesized and connected to the vector pDXA to construct the vector pDXAO3. Synthesize the Escherichia coli phosphoglucose isomerase gene promoter and tetracycline resistance gene, and link them into the vector pDXA to construct the vector pDXAPGI.

Example 3: Activity detection of the shuttle vector promoter in Escherichia coli

The shuttle vector pDXA was constructed by synthesizing the ampicillin resistance gene, promoter and pMB1 replicon sequence, and connecting them into the plasmid pDX of Lactobacillus acidophilus XW118. Tetracycline resistance genes connected to the promoters of ORF1, ORF2, ORF3 and ORF4 in the plasmid pDX were synthesized and connected to the vector pDXA to construct the vectors pDXAO1, pDXAO2, pDXAO3 and pDXAO4 respectively. The sequence of the plasmid vector pDXAO1 is shown in SEQ ID NO: 3; the sequence of the plasmid vector pDXAO2 is shown in SEQ ID NO: 4; the sequence of the plasmid vector pDXAO3 is shown in SEQ ID NO: 5; the sequence of the plasmid vector pDXAO4 The sequence is shown in SEQ ID NO:6. Synthesize the Escherichia coli phosphoglucose isomerase gene promoter and tetracycline resistance gene, and link them into the vector pDXA to construct the vector pDXAPGI. The shuttle vectors pDXAO1, pDXAO2, pDXAO3, pDXAO4 and pDXAPGI were respectively transformed into Escherichia coli ATCC25922, and cultured on LB plates containing ampicillin to obtain corresponding transformants. Spread Escherichia coli transformants on LB plates containing 50 μg/mL tetracycline, and culture them at 37°C, and observe whether there are colonies of transformants growing after cultivation. The results showed that colonies of Escherichia coli DXAO3, Escherichia coli DXAO4 and Escherichia coli DXAPGI grew, but no colonies of Escherichia coli DXAO1 and Escherichia coli DXAO2 grew. The promoters of the endogenous plasmids ORF3 and ORF4 of Lactobacillus acidophilus are active in Escherichia coli and can be used as Escherichia coli promoter elements for gene expression.

Example 4: Host range of shuttle vectors

In order to determine the host strain range of the Escherichia coli replicon Ori and the tetracycline resistance gene shuttle plasmid pDXAO4, the shuttle plasmid pDXAO4 was introduced into Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus plantarum, In the competence of Lactobacillus rhamnosus, Lactobacillus paracasei and Pediococcus lactis, lactic acid bacteria transformants were spread on MRS plates containing 50 μg/mL tetracycline, cultured at 30°C, and screened by resistance markers. The results showed that the shuttle plasmids could be electroporated in Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus paracasei, and obtained corresponding transformants, but No transformants were obtained in Pediococcus lactis.

Take 50, 200, and 500 ng of the shuttle vector pDXAO4 and mix them with various lactic acid bacteria in a competent state, activate them at 30°C for 3 hours after electric shock transformation, spread them on MRS plates containing 50 μg/mL tetracycline, and culture them at 30°C until the colonies grow long. out. Count the colonies of transformants and calculate the transformation efficiency (take the highest among the three gradients as the final transformation efficiency). Pick a single colony on the tetracycline-containing MRS plate and inoculate it into the tetracycline-containing MRS liquid medium, and incubate at 37°C for 24h. Then inoculate 1% inoculum into MRS liquid medium, culture at 37°C for 12 hours, and continue to subculture under this condition to 60 generations. The bacterial solution is diluted and spread on MRS solid plate containing tetracycline and MRS solid plate without tetracycline. plate, and calculate the loss rate of the plasmid according to the number of colonies on the two plates. Calculate the transformation efficiency and plasmid stability of the shuttle plasmid pDXAO4 in various lactic acid bacteria hosts. The results showed that the electrotransformation efficiency of the shuttle plasmid pDXAO4 was between 0.24×10 ² and 2.75×10 ³ CFU/μg (plasmid amount). The loss rate of the shuttle plasmid pDXAO4 was between 25.45% and 48.36%, among which the loss rate of the shuttle plasmid was 48.36% in Lactobacillus plantarum and 25.45% in Lactococcus lactis. The host range and transformation efficiency of the shuttle vector are shown in Table 1.

Table 1 Transformation efficiency and stability of the shuttle plasmid pDXAO4 in different hosts

Example 5: Induction of promoter by carbon source

Glucose, galactose, rhamnose, xylose, fructose and mannose were respectively used as carbon sources, and E. coli transformants containing pDXAO2 were cultured on medium containing 50 μg/mL tetracycline. The results showed that when glucose, rhamnose, xylose, fructose and mannose were cultured separately as carbon sources, no colonies of Escherichia coli DXAO2 grew. Escherichia coli DXAO2 colonies grew when galactose was used as carbon source.

Embodiment 6: lactic acid bacteria promoter is used for Escherichia coli expression enzyme gene

The phytase, amylase, protease and mannanase genes connected to the promoter of ORF4 in the plasmid pDX were respectively synthesized and connected to the vector pDXA to construct the vectors pDXA4P, pDXA4A, pDXA4R and pDXA4C respectively. The Escherichia coli phosphoglucose isomerase gene promoter and phytase, amylase, protease and mannanase genes were synthesized respectively, and respectively connected to the vector pDXA to construct the vectors pDXAPP, pDXAPA, pDXAPR and pDXAPC. The shuttle vectors pDXA4P, pDXA4A, pDXA4R, pDXA4C, pDXAPP, pDXAPA, pDXAPR and pDXAPC were transformed into Escherichia coli ATCC25922, respectively, and LB plates containing ampicillin were cultured to obtain corresponding transformants. The Escherichia coli transformants containing pDXA4P, pDXA4A, pDXA4R, pDXA4C, pDXAPP, pDXAPA, pDXAPR and pDXAPC were fermented and cultured for 24 hours, and the bacterial precipitates were collected, and the enzyme activities of phytase, amylase, protease and mannanase were measured respectively. The results showed that the E. coli pDXA4P phytase activity was 504U/mg, the E. coli pDXA4A amylase activity was 165U/mg, the E. coli pDXA4R protease activity was 387U/mg, and the E. coli pDXA4C mannanase activity was 243U/mg. pDXAPP phytase activity is 114U/mg, Escherichia coli pDXAPA amylase activity is 36U/mg, Escherichia coli pDXAPR protease activity is 52U/mg, Escherichia coli pDXAPC mannanase activity is 27U/mg, described phytase, The enzymatic activities of amylase, protease and mannanase are shown in Figure 4. It shows that the strength of the promoter of ORF4 is greater than that of E. coli phosphoglucose isomerase gene promoter in terms of enzyme gene expression.

The phytase gene sequence is shown in SEQ ID NO: 7; the mannanase gene sequence is shown in SEQ ID NO: 8; the protease gene sequence is shown in SEQ ID NO: 9; the amylase The gene sequence is shown in SEQ ID NO:10.

The phytase activity unit: the sample releases 1 μmol of inorganic phosphorus per hour from sodium phytate under the conditions of substrate sodium phytate concentration of 5.0 mmol/L, temperature of 37°C, and pH value of 5.5, which is one phytic acid Enzyme activity unit, expressed in U.

The amylase activity unit: the amount of enzyme required to catalyze the production of 1 μmol reducing sugar from substrate starch per hour under the conditions of substrate soluble starch concentration of 10 g/L, temperature of 37°C, and pH value of 6.0 is one activity The unit is represented by U.

The protease activity unit: when the sample is catalyzed to produce 1 μmol of tyrosine per hour from the substrate casein under the conditions of substrate casein concentration of 10 g/L, temperature of 37°C, and pH value of 7.0, it is a protease activity unit , represented by U.

The mannanase activity unit: the amount of enzyme required to produce 1 μmol of reducing sugar from the substrate per hour under the conditions of substrate mannan concentration of 3 g/L, temperature of 37°C, and pH value of 5.5 is an activity unit , represented by U.

Embodiment 7: lactic acid bacteria promoter is used for lactic acid bacteria expression enzyme gene

The phytase, amylase, protease and mannanase genes connected to the promoter of ORF4 in the plasmid pDX were synthesized respectively, and connected to the vector pDXAO3 to construct the vectors pDXA34P, pDXA34A, pDXA34R and pDXA34C respectively; The vectors pDXA34P, pDXA34A, pDXA34R and pDXA34C were transformed into Lactobacillus acidophilus, and the corresponding transformants were respectively obtained by coating the plates containing tetracycline. The transformants of Lactobacillus acidophilus containing pDXA34P, pDXA34A, pDXA34R and pDXA34C were fermented and cultured for 48 hours, the precipitates of Lactobacillus acidophilus were collected, and the enzyme activities of phytase, amylase, protease and mannanase were measured respectively. The results showed that the phytase activity of Lactobacillus acidophilus pDXA4P was 857U/mg, the activity of Lactobacillus acidophilus pDXA4A amylase was 1205U/mg, the activity of protease activity of Lactobacillus acidophilus pDXA4R was 792U/mg, and the activity of Lactobacillus acidophilus pDXA4C mannanase The activity is 517U/mg, and the enzyme activities of the phytase, amylase, protease and mannanase are shown in Table 2.

Table 2 Recombinant Lactobacillus acidophilus produces enzyme

Transform the vector pDXA34P into Lactococcus cremoris, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, respectively, and culture on plates containing tetracycline to obtain corresponding transformants . The lactic acid bacteria transformants containing pDXA34P were fermented and cultivated for 48h, and the enzyme activity of phytase was measured respectively. The results showed that different lactic acid bacteria transformants could produce phytase, and the Lactococcus lactis, Lactococcus lactis subsp. The enzyme activity of phytase produced by transformants of Bacillus, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus paracasei is shown in Table 3.

The phytase gene sequence is shown in SEQ ID NO: 7; the cellulase gene sequence is shown in SEQ ID NO: 8; the protease gene sequence is shown in SEQ ID NO: 9; the amylase The gene sequence is shown in SEQ ID NO:10.

The phytase activity unit: the sample releases 1 μmol of inorganic phosphorus per hour from sodium phytate under the conditions of substrate sodium phytate concentration of 5.0 mmol/L, temperature of 37°C, and pH value of 5.5, which is one phytic acid Enzyme activity unit, expressed in U.

The amylase activity unit: the amount of enzyme required to catalyze the production of 1 μmol reducing sugar from substrate starch per hour under the conditions of substrate soluble starch concentration of 10 g/L, temperature of 37°C, and pH value of 6.0 is one activity The unit is represented by U.

The protease activity unit: when the sample is catalyzed to produce 1 μmol of tyrosine per hour from the substrate casein under the conditions of substrate casein concentration of 10 g/L, temperature of 37°C, and pH value of 7.0, it is a protease activity unit , represented by U.

The mannanase activity unit: the amount of enzyme required to produce 1 μmol of reducing sugar from the substrate per hour under the conditions of substrate mannan concentration of 3 g/L, temperature of 37°C, and pH value of 5.5 is an activity unit , represented by U.

Table 3 Phytase produced by recombinant lactic acid bacteria

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included in the present invention. Within the protection scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims (3)

1. A shuttle vector pDXA of escherichia coli and lactobacillus, which is characterized in that the nucleotide sequence is shown in SEQ ID NO: 1.

2. The method of constructing a shuttle vector pDXA according to claim 1, comprising the steps of: the shuttle vector pDXA is constructed and obtained by synthesizing ampicillin resistance gene, promoter and pMB1 replicon sequence through genes and connecting the genes to lactobacillus acidophilus plasmid pDX; the plasmid pDX has a sequence shown in SEQ ID NO:2 is shown in the figure; the ampicillin resistance gene, the promoter and the pMB1 replicon sequence are shown in SEQ ID NO: 3.

3. Use of the shuttle vector pDXA according to claim 1 for the preparation of a pharmaceutical, food and feed additive.

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