CN106119221A - Phosphatidylinositol-specific phospholipase C gene, carrier, engineering bacteria and application - Google Patents

Abstract

The invention relates to a phosphatidylinositol-specific phospholipase C gene (its sequence is shown in SEQ ID No.1), a carrier, an engineering bacterium, and a medicament for preparing anti-chicken coccidia that can be expressed heterologously by microorganisms in the application. The beneficial effects of the present invention are mainly reflected in: the present invention optimizes the design and synthesis of the PI-PLC target gene, successfully realizes the heterologous high expression of the PI-PLC gene in Escherichia coli, and the recombinant protein shows a higher activity of phospholipase, and the expressed PI‑PLC was used in animal experiments against chicken coccidia, and the anti-coccidia effect was good.

Description

Phosphatidylinositol-specific phospholipase C gene, carrier, engineering bacteria and application

(1) Technical field

The invention relates to a phosphatidylinositol-specific phospholipase C gene, a carrier, an engineering bacterium, and an application thereof in preparing medicines against chicken coccidia.

(2) Background technology

There are many types of phosphatidylinositol-specific phospholipase C (PI-PLC), which exist in bacteria, protozoa, yeast, mold, plants, insects and mammals. There are applications in various fields: in the field of medicine, it is mainly used as a vaccine to prevent infection of various pathogenic bacteria; in the field of food, it is mostly used in bread baking, dairy product processing, health food, etc.; properties, animal feed additives, etc.

The chicken coccidiosis incidence rate is high (50-70%), and the lethality rate is high (50-80%), which seriously endangers the aquaculture industry, and the loss caused by coccidiosis is as high as billions of dollars every year. Existing prevention and treatment of chicken coccidiosis mainly relies on chemical drugs, but there are deficiencies such as drug resistance and drug residues. If vaccination is used for prevention and control, there are also disadvantages of high risk and difficulty in research and development. Using antibiotics or Chinese herbal medicines, There is also the problem of drug resistance, and the effect is average. Therefore, there is an urgent need to find a new anticoccidial drug to replace chemical drugs to effectively control chicken coccidiosis.

Bacterial PI-PLC can cleave the glycosyl-anchored protein on the cell membrane surface, thereby affecting the release of glycoproteins and carbohydrates on the cell membrane surface, while most pathogenic parasite cell membrane surface antigens such as coccidia are anchored on the cell surface through glycosyl groups. On the above, PI-PLC can cut off the connection between the inositol phospholipid in the anchor protein and the cell membrane, so that the parasite loses the ability to invade and proliferate in the host cell, so PI-PLC shows significant anti-coccidia infection characteristics.

The anticoccidial mechanism of PI-PLC provides a theoretical basis for the development of new anticoccidial drugs. PI-PLC reduces the risk of outbreaks of coccidia due to increased drug resistance, and avoids major losses caused by relapse of coccidiosis in the breeding industry. PI-PLC is widely distributed in the body of animals, plants and microorganisms. It itself participates in the metabolism and information exchange of body cells, and there are no problems such as drug resistance and drug residues. It is safe and non-toxic. Through the heterologous expression of microorganisms, PI-PLC can be produced in large quantities, and the cost of using anticoccidiostats can be reduced.

(3) Contents of the invention

The object of the present invention is to provide a phosphatidylinositol-specific phospholipase C gene, a carrier, an engineering bacterium that can be expressed heterologously by microorganisms, and its application in the preparation of anti-coccidian medicaments.

The technical scheme adopted in the present invention is:

Phosphatidylinositol-specific phospholipase C gene, the sequence of which is shown in SEQ ID No.1. The sequence is a Bacillus cereus PI-PLC gene (Genbank: M30809.1) obtained from the NCBI database. According to the codon preference of Escherichia coli and under the premise of keeping the amino acid sequence unchanged, it was optimized and synthesized. The PI-PLC target gene is easy to be stably expressed in Escherichia coli and has a high expression level.

The sequence of SEQ ID No.1 is as follows:

5’-ATGTCTAACAAAAAACTTATCCTGAAATTATTTATCTGCTCCACAATCTTTATTACATTTGTCTTTGCTCTGCATGACAAACGGGTGGTTGCAGCGTCAAGCGTGAACGAACTGGAAAATTGGAGCAAATGGATGCAACCGATTCCTGATTCAATCCCGCTTGCGCGTATTAGCATCCCTGGCACGCATGACTCTGGAACATTTAAATTGCAAAACCCGATCAAACAGGTTTGGGGCATGACACAAGAATACGATTTTCGTTATCAGATGGACCATGGTGCTCGGATTTTTGATATCAGAGGCCGCTTAACGGATGACAATACAATCGTTTTGCATCATGGACCGTTGTACTTGTACGTGACGTTGCATGAATTTATCAACGAAGCGAAACAGTTTTTGAAAGATAACCCTTCAGAAACAATCATCATGAGCTTGAAAAAAGAATACGAAGATATGAAAGGCGCTGAAGACTCTTTTTCTTCCACGTTTGAGAAAAAATACTTTGTCGATCCTATCTTTCTGAAAACAGAAGGAAACATCAAACTTGGAGACGCCAGAGGTAAAATTGTACTGCTTAAACGCTATTCTGGTTCCAACGAACCGGGCGGATACAACAACTTTTACTGGCCTGATAACGAAACGTTTACAACGACAGTGAATCAAAACGCAAATGTTACAGTGCAGGATAAATACAAAGTCTCCTACGACGAAAAAGTAAAAAGCATCAAAGATACGATGGACGAAACAATGAATAACTCTGAAGATCTGAACCATCTTTACATCAACTTTACGTCACTTTCAAGCGGTGGCACAGCTTGGAATTCTCCGTATTACTATGCCTCCTACATCAACCCTGAAATCGCAAACTACATCAAACAGAAAAATCCGGCACGCGTCGGATGGGTAATCCAGGATTATATTAATGAAAAATGGAGCCCGTTACTGTATCAAGAAGTCATCAGAGCAAATAAATCCTTAATCAAAGAATAA-3’。

The invention also relates to a recombinant vector containing the phosphatidylinositol-specific phospholipase C gene.

The invention also relates to the recombinant genetically engineered bacteria transformed by the recombinant vector.

The present invention also relates to the application of the phosphatidylinositol-specific phospholipase C gene in the preparation of anti-chicken coccidia medicine.

Specifically, the application is: constructing a recombinant vector containing the phosphatidylinositol-specific phospholipase C gene, transforming the recombinant vector into Escherichia coli, obtaining recombinant genetically engineered bacteria for induction culture, and separating the culture medium A supernatant containing phosphatidylinositol-specific phospholipase C was obtained.

Through the initial optimization of the culture conditions of recombinant E. coli shake flasks, with 4% transfer amount, 37 ° C, 200r/min conditions, when culturing recombinant E. coli OD600 = 0.4, add the inducer IPTG, the working concentration is 1mM , to induce expression, and after continuing to culture at 37°C and 200r/min for 6h, the concentration of PI-PLC in the broken supernatant of the culture medium was measured to be above 10mg/L.

The beneficial effects of the present invention are mainly reflected in: the present invention optimizes the design and synthesis of the PI-PLC target gene, successfully realizes the heterologous high expression of the PI-PLC gene in Escherichia coli, and the recombinant protein shows a higher activity of phospholipase, and the expressed PI-PLC was used in animal experiments against chicken coccidia, and the anti-coccidia effect was good.

(4) Description of drawings

Figure 1 is the amplification result of the target gene; M: DNA Maker; 1: PCR amplification product 1; 2: PCR amplification product 2;

Figure 2 is a structural map of the plasmid pET28a(+);

Fig. 3 is the construction process of recombinant plasmid pET28a (+)-PIPLC;

Figure 4 is a single bacterium colony in the recombinant escherichia coli;

Fig. 5 is the colony PCR result figure of recombinant escherichia coli;

Figure 6 is the result of SDS-PAGE electrophoresis identification of recombinant protein; M: standard protein Maker; 1: broken supernatant of pET28a(+)/BL21 control bacteria; 2: broken supernatant of induced pET28a(+)-PIPLC/BL21;

Figure 7 shows the results of PI-PLC enzyme activity verification (PI color plate); 1: the broken supernatant of recombinant bacteria pET28a(+)-PIPLC/BL21; 2: the broken supernatant of recombinant bacteria pET28a(+)/BL21 (CK);

Fig. 8 is an enzyme-linked reaction analysis and determination enzyme content result figure;

Figure 9 is a comparison of the body weight changes of different experimental groups;

Figure 10 is the comparison of the number of coccidia per gram of feces (22 days OPG count);

Figure 11 is the comparison of the anti-coccidian index ACI of different experimental groups.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1:

1. Optimization and synthesis of the target gene

A PI-PLC gene of Bacillus cereus obtained from the NCBI database (Genbank: M30809.1), according to the codon preference of Escherichia coli, under the premise of keeping the amino acid sequence unchanged, the PI-PLC was optimized and synthesized The target gene (SEQ ID No.1) was synthesized by Hangzhou Qingke Zixi Biotechnology Co., Ltd.

2. Acquisition and identification of target gene fragments

Specific steps: design primers based on the synthesized known sequences. Forward primer P1: 5’-CCCGGTCTCCCATGGGCTCTAACAAAAAACTTATCCTGAAA-3’, reverse primer P2: 5’-CCCGGTCTCGAATTCTTATTCTTTTGATTAAGGATT-3’, the underline represents the restriction site containing BsaI. The synthetic gene PI-PLC was used as template and p1/p2 as primers for PCR amplification.

The PCR amplification system is: 1 μL of template, 1 μL of upstream and downstream primers, 25 μL of 2×super HIFI-MIXⅡ, 20 μL of sterilized double distilled water. The PCR amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 60 s, and extension at 72°C for 10 min after 32 cycles. PCR products were identified by 1% agarose gel electrophoresis analysis. After digestion with the endonuclease BsaI, the sticky ends of the NcoI restriction site and the EcoRI restriction site are generated, and the PCR product is recovered to obtain the target gene.

See Figure 1 for the amplification results of the target gene, and Figure 2 for the structural map of the plasmid pET28a(+).

3. Construction of recombinant plasmid pET28a(+)-PIPLC

Specific steps: Recover the target gene by PCR, digest it with BsaI, connect it with pET28a digested with EcoRI and NcoII, and transform it into the cloning host bacteria, then select PIPLC primers to amplify, pick out positive clones for sequencing, and correctly recombine the plasmid Named as pET28a(+)-PIPLC, the obtained recombinant Escherichia coli was named Trans1-T1-PIPLC/Trans1-T1, and stored in a glycerol tube.

See Figure 3 for the construction process of the recombinant plasmid pET28a(+)-PIPLC.

4. Cloning of recombinant plasmid pET28a(+)-PIPLC in Escherichia coli

Specific steps: extract the correct recombinant plasmid pET28a(+)-PIPLC, transfer it to the expression host bacterium BL21(DE3) by the method of general chemical transformation, pick the transformant and perform colony PCR with P1 and P2 primers, pick and amplify the target The transformants of size fragments were inoculated in LB (containing 50 μg/mL kanamycin) liquid medium for expanded culture, and cultured overnight at 37°C. The obtained recombinant bacteria were named pET28a-PIPLC/BL21(DE3), and stored in glycerol tubes.

See Figure 4 for the single colony diagram in recombinant Escherichia coli, and Figure 5 for the colony PCR result diagram of recombinant Escherichia coli.

5. Identification of recombinant protein by SDS-PAGE electrophoresis

Specific steps: Pick a single colony of positive recombinant bacteria pET28a-PIPLC/BL21(DE3) and inoculate it in 5ml LB (containing 1 μg/mL kanamycin) liquid medium, and culture it overnight at 37°C for 20 hours, then carry out at a ratio of 1:50 Expand the culture to OD value = 0.3-0.6 and add the inducer IPTG with a final concentration of 0.75mM for induction for 4 hours. Collect the cells, add 1/20 volume of cell buffer NTA-0Buffer (20mM Tris-HCl pH7.9, 0.5M NaCl, 10% Glycerol)

Take 50ul of the bacterial solution and add the sample buffer to mix and boil for 10min. After cooling, take 20μL for SDS-PAGE (12% separating gel, 5% stacking gel) electrophoresis analysis, with the standard molecular weight of the protein as a reference, and treat in the same way The pET28a-PIPLC/BL21(DE3) strain without inducer was used as a control.

See Figure 6 for the electrophoresis results. It can be seen from the figure that a fragment with a size of about 35 kDa appeared in the crushed supernatant of the induced recombinant strain pET28a(+)-PIPLC/BL21, which was consistent with the size of PI-PLC. 6. PI-PLC enzyme activity verification

Specific steps: Take 10 μL of recombinant bacteria pET28a-PIPLC/BL21(DE3) cultured for 12 hours on the PI-Listeria chromogenic plate, and treat pET28a/BL21(DE3) blank bacteria as a control in the same way, 37 Incubate at ℃ for 12h. Results: The recombinant pET28a-PIPLC/BL21(DE3) showed a transparent circle on the plate, but there was no transparent circle in the control. The results are shown in Figure 7, which shows that the recombinant strain pET28a(+)-PIPLC/BL21 has phospholipase activity.

7. Quantitative determination of PI-PLC content in bacterial liquid by enzyme-linked reaction

Specific steps:

① Treatment of samples to be tested: place the recombinant Escherichia coli liquid after induced expression on ice and ultrasonically crush until clarified, centrifuge at 8000r/min for 10min, and collect the supernatant;

②Dilution of the standard product: The kit provides a PI-PLC standard product at original times, which should be diluted in a 1.5mL centrifuge tube according to the chart below.

③ Adding samples: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and sample wells to be tested. Accurately add 50 μL of the standard substance on the enzyme-labeled plate, add 40 μL of the sample diluent to the well of the sample to be tested, and then add 10 μL of the sample to be tested (the final dilution of the sample is 5 times). Adding the sample Add the sample to the bottom of the well of the microtiter plate, try not to touch the wall of the well, shake gently to mix.

④Incubation: Seal the plate with a sealing film and incubate at 37°C for 30min.

⑤ Dosing: Dilute the 30-fold concentrated washing solution 30-fold with distilled water for later use.

⑥Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

⑦Enzyme addition: Add 50 μL of enzyme-labeled reagent to each well, except for blank wells.

⑧Incubation: The operation is the same as 3.

⑨ Washing: The operation is the same as 5.

⑩Color development: first add 50 μL of chromogenic reagent A to each well, then add 50 μL of chromogenic reagent B, shake and mix gently, and develop color at 37°C in the dark for 15 minutes.

Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color turns yellow immediately).

Determination: Measure the absorbance (OD value) of each well sequentially with a blank air conditioner and a wavelength of 450nm. The measurement should be carried out within 15 minutes after adding the stop solution.

The calculation takes the concentration of the standard as the abscissa, and the OD value as the ordinate, draws the standard curve on the coordinate paper, and finds out the corresponding concentration from the standard curve according to the OD value of the sample; then multiplies it by the dilution factor; or uses the OD value of the standard. Concentration and OD value calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor, which is the actual concentration of the sample.

The results are shown in Figure 8, which shows that Escherichia coli was induced to express PI-PLC according to the above method, and the concentration of PI-PLC in the culture solution (supernatant) after induction was finally measured to be 10 mg/L.

Example 2:

In order to study the effect of bacteria PI-PLC on chicks’ anti-coccidial and weight gain effects, the most pathogenic Eimeria tenella was selected to infect normal chicks, and grouped chicks were given different drug treatments. Observe and record the clinical pathological changes, survival rate, body weight change, cecal lesion and oocyst value of chicks, so as to calculate the anticoccidial index of PI-PLC.

1. Experimental group

Detection Indicator:

(1) Chick weight

(2) Scoring of cecal lesions

(3) Oocyst value

(4) Anti-coccidia index (ACI)

2. Experimental results

1. Pathological observation

Four days after the normal chicks were infected with coccidia, they were depressed, their appetite decreased, and their feathers were fluffy.

Normal chicks were infected with coccidia on the 5th day, severe cases died, dissected, and the cecum was congested and enlarged, which was a typical coccidia fatality.

2. Weight change

See Figure 9 for the body weight change data of each experimental group. It can be seen from the figure: the growth of the negative control group was the slowest, and one chick died, indicating that coccidiosis seriously affected the normal growth and development of chickens, and severe cases could lead to death; The blank control group of coccidia was still higher, and the relative weight gain rate reached 102.32%, which also explains why diclazuril is widely used in the breeding industry as an anticoccidial drug; the PI-PLC experimental group The relative weight gain rate of chicks was higher than that of negative control group and PI-PLC control group, indicating that PI-PLC had a certain effect on the weight gain of chicks infected with coccidia.

3. Scoring of cecal lesions

The scoring data of cecal lesions in each experimental group are shown in the table below:

group Cecal lesion score lesion value Blank control group 0.0 0 negative control group 3.25 32.5 Diclazuril group 1.5 15 PI-PLC experimental group 1.75 17.5 PI-PLC control group 2.85 28.5

Note: Cecal lesions are scored in five grades of 0, 1, 2, 3, and 4. The higher the score, the more serious the cecal lesions.

It can be seen from the table that the cecal lesion scores of the chicks in the diclazuril group and the PI-PLC experimental group were significantly lower than those in the negative control group and the PI-PLC control group, indicating that these two groups have a significant effect against coccidiosis.

4. The number of coccidia per gram of feces (OPG count)

See Figure 10 for the results of the number of coccidia per gram of feces in each experimental group. It can be seen from the figure that although the oocyst ratio of the PI-PLC group is much higher than that of the diclazuril group, it is still significantly lower than the two control groups, indicating that PI-PLC, like diclazuril, had an obvious inhibitory effect on the growth and reproduction of coccidia.

5. Anti-coccidial index (ACI)

See Figure 11 for the results of the anticoccidial (ACI) index data of each experimental group. Anti-coccidial index ACI = (chicken survival rate + relative weight gain rate) × 100 - (oocyst value + lesion value).

The ACI index shows that the anticoccidial index of the diclazuril group is the highest, reaching over 180, which can effectively promote the weight gain of chicks, and inhibit the growth of coccidia in the cecum of chicks, and the anticoccidial effect is at an excellent level; the PI-PLC group Although the ACI index was lower than that of the diclazuril group, it was significantly higher than that of the negative control group and the PI-PLC control group. The ACI index was close to 160, indicating that PI-PLC had an anti-coccidia effect, and the anti-coccidia effect was close to good.

3. Conclusion

The experimental results showed that the anti-coccidiostat index ACI of bacterial PI-PLC was close to 160, indicating that PI-PLC had an anti-coccidiostat effect, and the anti-coccidia effect was close to good.

Claims (5)

1. phosphatidylinositol-specific phospholipase C gene, its sequence is as shown in SEQ ID No.1.

2. contain the recombinant vector of phosphatidylinositol-specific phospholipase C gene described in claim 1.

3. utilize recombinant vector described in claim 2 to convert the recombination engineering bacteria obtained.

4. the answering in the medicine preparing anti-Eimeria species of described phosphatidylinositol-specific phospholipase C gene described in claim 1 With.

Apply the most as claimed in claim 4, it is characterised in that described application is: build containing described phosphatidylinositols special Property phospholipase C gene recombinant vector, described recombinant vector is converted in escherichia coli, it is thus achieved that recombination engineering bacteria is carried out Inducing culture, culture fluid isolated contains the supernatant of phosphatidylinositol-specific phospholipase C.