CN105200028A - Endolysin derived from Vibrio parahaemolyticus phage and its application - Google Patents

Abstract

The invention belongs to the field of biotechnology, and particularly relates to an endolysin derived from the Vibrio parahaemolyticus phage, and application of the endolysin. The endolysin is derived from the Vibrio parahaemolyticus phage of which the amino acid sequence is all or a part of the shown amino acid sequence SEQ ID No: 1. Sequence analysis shows that the endolysin is a lyase in the phase, and the lyase enables a bacterial cell wall to be cracked; test confirms that protein formed from the amino acid sequence is better in bactericidal activity. The endolysin has an obvious bactericidal effect on in vitro Vibrio parahaemolyticus after being expressed and purified by a suitable expression vector existing in the market, and can be applied to preparation of a Vibrio parahaemolyticus bacteriostat.

Description

Endolysin derived from Vibrio parahaemolyticus phage and its application

technical field

The invention belongs to the field of biological technology, and in particular relates to an endolysin derived from Vibrio parahaemolyticus phage and application thereof.

Background technique

Vibrio parahaemolyticus is a Gram-negative polymorphic bacillus or slightly curved Vibrio, which is a secondary harmful microorganism and belongs to the genus Vibrio in the Vibrio family. Seawater, seabed sediments, and seafood such as fish and shellfish. With the extensive and long-term use of antibiotics, the increasingly serious drug-resistant Vibrio parahaemolyticus in food and the environment has affected the treatment of human infection with Vibrio parahaemolyticus. Can new biological antibacterial agents be developed to replace chemical Antibiotics are currently a challenge for researchers.

Phage endolysins are a class of lytic enzymes capable of cleaving bacterial cell walls. Although the ability of phage endolysin to lyse bacteria was first reported in 1957, it was not until 2001 that Nelson et al. confirmed that purified recombinant endolysin could be used as an antibacterial agent to effectively control group A streptococcus infection in rats. So far, endolysins have been used to control and detect foodborne pathogens such as Staphylococcus aureus, Listeria and Clostridium in food. Compared with the use of small molecule antibiotics for antibacterial treatment or prevention, specific endolysins are less likely to lead to the rapid emergence of resistant strains. Now that bacterial drug resistance is becoming more and more serious, especially in the unprecedented market demand for new antibacterial agents, endolysin is not easy to produce resistance and has the characteristics of cleavage specificity, which makes it have certain advantages as a new type of antibacterial agent.

Although the domestic use of genetic engineering technology to construct endolysin-producing strains has made some progress, an important problem in the research and production process is that most of the prepared endolysins are concentrated in Gram-positive bacteria. Prevention and detection of Gram-negative bacteria, especially Vibrio parahaemolyticus, is still less researched. Therefore, in view of the increasing drug resistance of Vibrio parahaemolyticus and the lack of related endolysins, the present invention develops endolysins or endolysin genes capable of efficiently lysing Vibrio parahaemolyticus.

Contents of the invention

The invention provides an endolysin from Vibrio parahaemolyticus phage with significant antibacterial effect.

The present invention also provides the application of the endolysin in the preparation of antibacterial agents.

The technical solution adopted by the present invention to solve its technical problems is:

A phage with environmental disinfection ability, the phage monomer is Vibrio parahaemolyticus phage, the Latin name is Vibrioparahaemolyticusphage, named qdvp001, the preservation number: CCTCCNO: M2011143, and the preservation date is April 26, 2011. The vibrio parahaemolyticus phage qdvp001 is a potent phage and has the function of lysing vibrio parahaemolyticus.

The phage has a strong lytic effect on Vibrio parahaemolyticus, and provides a source of phage for industrial production of phage and for disinfection and sterilization.

An endolysin derived from the above-mentioned Vibrio parahaemolyticus phage, the amino acid sequence of which is all or part of the amino acid sequence shown in SEQ ID No: 1. Sequence structure analysis shows that the enzyme is a lytic enzyme capable of cleaving bacterial cell walls in bacteriophage, and it is confirmed by experiments that the protein formed by the amino acid sequence has better bactericidal activity.

The gene encoding the endolysin, the nucleotide sequence of the gene is shown in SEQ ID No: 2.

Expression vectors, engineering bacteria or cell lines containing the genes.

The prokaryotic cell expression vector is pET-30a(+).

The engineering bacteria is Escherichia coli Rosseta (DE3).

An application of the endolysin in the preparation of antibacterial agents. At present, there is no document disclosing that phage qdvp001 of Vibrio parahaemolyticus can produce a lysing enzyme that can crack the cell wall of Vibrio parahaemolyticus, but the endolysin of the present invention has this function and has effective bactericidal activity against Vibrio parahaemolyticus.

The beneficial effects of the present invention are: after the endolysin of the present invention is expressed and purified through a suitable expression vector existing on the market, it exhibits a significant bactericidal effect on Vibrio parahaemolyticus in vitro, and can be used as a bacteriostatic agent for Vibrio parahaemolyticus. preparation.

Description of drawings

Fig. 1 is the domain analysis prediction diagram of endolysin protein;

Figure 2 is the high-efficiency expression map of endolysin protein in Escherichia coli, M is marker, 1 is the total bacterial protein not induced by IPTG, 2, 3, 4, and 5 eluate collected during column passing, 6 is nickel The fusion protein after column purification, 7 is the fusion protein after concentration and desalination by ultrafiltration, and 8 is the induced bacterial total protein;

Fig. 3 is the change spectrum of the absorbance value of Vibrio parahaemolyticus ATCC17802 by endolysin protein cleavage;

Fig. 4 is a diagram of the turbidity change of Vibrio parahaemolyticus ATCC17802 lysed by endolysin protein.

Detailed ways

The technical solution of the present invention will be further specifically described below through specific examples. It should be understood that the implementation of the present invention is not limited to the following examples, and any modifications and/or changes made to the present invention will fall within the protection scope of the present invention.

In the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or commonly used in the field. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

Bacterial strains involved in the present invention's test: Vibrio parahaemolyticus VP17802 (preservation number is ATCC17802), purchased from U.S. ATCC Center, Vibrio parahaemolyticus VIB304, Vibrio parahaemolyticus VIB461, Vibrio parahaemolyticus VIB800 and Vibrio parahaemolyticus VP14-90 , VP1.2, VP2.1, VP3.2, and VP4.1 are all stored in the Food Safety Laboratory of Ocean University of China.

The Vibrio parahaemolyticus phage involved in the present invention, the phage qdvp001 is preserved in the China Type Culture Collection Center, address: Wuhan, China, Wuhan University, preservation number: CCTCCNO: M2011143, preservation date is April 26, 2011, taxonomic name : Vibrio parahaemolyticus phage qdvp001 (Vibrioparahaemolyticusphageqdvp001).

2216E culture medium, Qingdao Haibo Biotechnology Co., Ltd.;

LB liquid medium, Beijing Land Bridge Technology Co., Ltd.;

Plasmid pET30a and Escherichia coli Rosetta (DE3), Beijing Kangwei Century Company;

NiSepharose ^TM 6FastFlow filler, American GE Company;

Isopropyl-β-D-thiogalactopyranoside (IPTG), Beijing Suo Laibao Technology Co., Ltd.

Example 1: Prediction of endolysin protein function

The present inventor isolated a virulent phage qdvp001 of Vibrio parahaemolyticus from sewage. After whole genome sequencing and analysis, it was identified that the protein encoded by the phage ORF60 had 56% similarity in amino acid sequence with the substrate and tail hydrolase of Vibrio phage ICP1. Domain prediction analysis of ORF60-encoded protein Lysqdvp001 was performed using SMART software. The domain prediction results are shown in Figure 1. The 9-65 amino acid range of Lysqdvp001 is a highly conserved functional region and belongs to the PG_binding (PF01471) domain, which is related to cell wall peptidoglycan binding. The 123-216 amino acid interval of Lysqdvp001 is a highly conserved functional region, which belongs to the CHAP family (PF05257), which is related to the hydrolysis of prokaryotic cell wall peptidoglycan.

Example 2: High expression of endolysin in Escherichia coli

1. Construction of recombinant plasmids

According to the endolysin gene sequence (SEQ ID No: 2), primers were designed, upstream primer: CGGGATCCATGACTTTAATTCGTAAGGGTAGTCG (SEQ ID No: 3), downstream primer: CCGCTCGAGTTAAGCTTCGTTATTACTAGTTACATCTGA (SEQ ID No: 4), restriction enzymes BamHI and XhoI. The endolysin gene was amplified by PCR, and 25 μL of the PCR recovered product was cloned into the multiple cloning site BamHI and XhoI of the pET-30a vector to obtain a recombinant plasmid, which was transformed into Escherichia coli Rosetta (DE3). Single clones were picked and cultured in LB liquid medium with shaking at 37°C for 5 hours, and then sent to Shanghai Sangong for sequencing. Sequencing results showed that the plasmid had the nucleotide sequence shown in SEQIDNo: 2, and the protein expressed by the gene was named Lysqdvp001, and the gene encoded the amino acid residue sequence shown in SEQIDNo: 1. The correctly identified recombinant plasmid was named pET-30a-Lysqdvp001.

2. Preparation of recombinant protein

The recombinant plasmid named pET-30a-Lysqdvp001 was transformed into Escherichia coli Rosetta (DE3), and the engineered strain Rosetta (Lysqdvp001) capable of expressing endolysin was screened, and a single colony was inoculated in 5 mL LB liquid medium (containing 50 μg/mL kanamycin) Medium, 37°C, 150r/min, overnight shaking culture, the next day, according to the ratio of 1:50, add the bacterial solution into fresh 300mL LB liquid medium (containing 50μg/mL kanamycin), and cultivate to OD at 37°C _600nm 0.6, add IPTG (to a final concentration of 1mmol/L), induce expression at 37°C, shake at 150r/min for 4h, centrifuge at 3381g, 4°C for 15min to obtain cells. Suspend the cell pellet obtained from 300mL bacterial solution in 30mL buffer solution (20mmol/LpH8.0NaH ₂ PO ₃ , containing 500mmol/LNaCl), mix well, break up with an ultrasonic breaker, and centrifuge to remove insoluble cell fragments. 0.22μm sterile filter membrane to obtain crude enzyme solution. NiSepharose ^TM 6FastFlow was used for purification, and a 10kD ultrafiltration centrifuge tube was used for desalting treatment to obtain recombinant protein with high purity. The results are shown in Figure 2, the recombinant protein Lysqdvp001 was highly expressed. In Figure 2, M is the marker, 1 is the total bacterial protein that has not been induced by IPTG, 2, 3, 4, and 5 are the eluents collected during the column passage, 6 is the fusion protein purified by the nickel column, and 7 is the ultrafiltration concentration The fusion protein after desalting, 8 is the total bacterial protein induced

Example 3: Taking Vibrio parahaemolyticus ATCC17802 as the target bacterium to test the antibacterial effect of endolysin LysVPp1

Pick a single colony of Vibrio parahaemolyticus ATCC17802 into 300mL2216E medium, culture overnight, centrifuge the bacteria, reconstitute with 100mM EDTA for 5min, wash the cells twice with pure water, and then store them at -80°C. Before measuring the activity, the bacteria The precipitate was reconstituted with 50mmol/LpH8.2 Tris containing 0.1% TritonX-100. Add 100 μL of recombinant protein Lysqdvp001 solution (400 μg/mL) to 100 μL of bacterial reconstitution solution, and mix buffer and lysozyme (400 μg/mL) with the bacterial reconstitution solution as blank group and positive control group respectively, and culture under the same conditions , Measure the OD450 value with a microplate reader, and the decrease in absorbance reflects that the bacteria are lysed.

The results are shown in Figure 3, the absorbance of the recombinant protein Lysqdvp001 test group and the positive control group decreased rapidly within 5 minutes, and the Lysqdvp001 solution reduced the bacterial turbidity by about 0.66 within 15 minutes, and the lysozyme solution reduced by about 0.66. As shown in Figure 4, after incubation at 37°C for 15 minutes, the bacterial solution of the Lysqdvp001 test group and positive control group was significantly clearer than that of the blank group. It was proved that the endolysin Lysqdvp001 obtained by cloning expression had antibacterial activity in vitro.

Example 4: Determination of the antibacterial effect of endolysin LysVPp1 with different serotypes of Vibrio parahaemolyticus and Escherichia coli as target bacteria

Pick a single colony of Vibrio parahaemolyticus ATCC17802 into 300mL2216E medium, culture overnight, centrifuge the bacteria, reconstitute with 100mM EDTA for 5min, wash the cells twice with pure water, and then store them at -80°C. Before measuring the activity, the bacteria The precipitate was reconstituted with 50mmol/L pH8.0 Tris containing 0.1% TritonX-100. Add 100 μL of recombinant protein Lysqdvp001 solution (400 μg/mL) to 100 μL of bacterial reconstitution solution, and measure the OD450 value. Lysqdvp001 enzyme activity value = Δ450nm (reduced value of Lysqdvp001 experimental group) - Δ450nm (reduced value of buffer blank group).

Using Vibrio parahaemolyticus ATCC17802 as the antibacterial positive standard, the antibacterial activity of Lysqdvp001 against multiple strains was detected. As shown in Table 1, Lysqdvp001 can lyse the remaining five different serotypes of Vibrio parahaemolyticus.

Table 1 Analysis of antibacterial effect of endolysin Lysqdvp001

The embodiment described above is only a preferred solution of the present invention, and does not limit the present invention in any form. There are other variations and modifications on the premise of not exceeding the technical solution described in the claims.

Claims (8)

1. derive from an endolysin for vibrio parahaemolyticus phage, its aminoacid sequence is all or part of aminoacid sequence shown in SEQIDNo:1.

2. the gene of coding endolysin according to claim 1, the nucleotide sequence of described gene is as shown in SEQIDNo:2.

3. the expression vector containing gene according to claim 2, engineering bacteria or clone.

4. expression vector according to claim 3, is characterized in that: described procaryotic cell expression carrier is pET-30a (+).

5. engineering bacteria according to claim 3, is characterized in that: described engineering bacteria is intestinal bacteria Rosseta (DE3).

6. an endolysin according to claim 1 is preparing the application in fungistat.

7. one kind has the phage of environment disinfected ability, it is characterized in that: this phage monomer is vibrio parahaemolyticus phage, and Latin is called Vibrioparahaemolyticusphage, is named as qdvp001, deposit number: CCTCCNO:M2011143, preservation date is on April 26th, 2011.

8. the phage with environment disinfected ability according to claim 7, is characterized in that: this phage has strong splitting action to Vibrio parahaemolyticus, provides phage source for suitability for industrialized production phage and for disinfection.