CN118146322B - Lawsonia intracellularis flgE protein, and preparation method, gene, application and kit thereof - Google Patents

Abstract

The invention belongs to the field of biology, and discloses lawsonia intracellularis flgE protein, wherein the amino acid sequence of the flgE protein is shown as SEQ ID No. 2. Based on the structural analysis of flgE protein in the early stage, more than ten different flgE truncated sequences are designed, 3 experiments are optimized, and a group of flgE truncated sequences with better effects are obtained. The protein expressed by the sequence protein has better specificity, sensitivity, repeatability and relativity to Lawsonia intracellularis after being applied to the kit. Meanwhile, the invention also discloses a preparation method, genes, application and a kit of the protein.

Description

Lawsonia intracellularis flgE protein, and preparation method, gene, application and kit thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to lawsonia intracellularis flgE protein, a preparation method, a gene, application and a kit thereof.

Background

The proliferative enteritis (proliferative enteropathy, PE) is a contact infectious disease caused by Lawsonia intracellularis (lawsonia intracellularis, LI), can cause lower death rate, but can cause slow growth of pig groups, prolonged time for marketing, severely reduced feed return rate, poor carcass uniformity during marketing, high infection rate of growing pigs, high proportion of invisible infection cases, easy neglect, and great economic loss for pig industry. At present, the laboratory diagnosis can be carried out by pathological tissue examination, such as a Warthin-Starry silver staining method or a modified Ziehl-Neelsen method, and the intestinal glandular hyperplasia condition, cytopathy and the presence or absence of pathogenic bacteria in cells are observed; indirect Immunofluorescence (IFA) or Immunohistochemistry (IHC) can also be used, but this only detects post-mortem cases and does not detect activity. The detection method of Polymerase Chain Reaction (PCR) belongs to the detection category of molecular biology, and has high sensitivity, but the activity of pathogenic bacteria cannot be determined. ELISA method can detect whether the active serum contains antibody or the rule of the antibody to judge the infection or immunity of pig group.

Several proteins of L.intracellularis such as flagellin flgE, LI0710, autotransporter LI0649, chaperonin HSP60, outer membrane protein Omp2, OMP1022, OMP0902, OMP1024, and surface lipoprotein SLP0235 have been reported after antigen or immunogenicity, wherein few proteins are reported to be expressed and then used as target antigens, and ELISA detection methods are established to detect L.intracellularis antibodies in pigs, but no mature and widely circulated commercial kit exists in China at present. And the commercialized kit imported abroad is high in price, long in purchasing period and unfavorable for clinical detection and use.

As for the detection methods of L.intracellularis, CN202110135312.3 and CN202311229730.4 are available, and both schemes are to amplify the full-length fragment of flgE gene and to express the protein by prokaryotes, and proved by verification, if the kit is prepared from the protein expressed by the full-length fragment of the original flgE gene, the kit has disadvantages in terms of specificity, sensitivity and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide lawsonia intracellularis flgE protein, designs more than ten different flgE truncated sequences based on the structural analysis of flgE protein in the earlier stage, and performs experiments on 3 preferred types of the flgE truncated sequences to obtain a group of flgE truncated sequences with better effects. The protein expressed by the sequence protein has better specificity, sensitivity, repeatability and relativity to Lawsonia intracellularis after being applied to the kit.

Meanwhile, the invention also discloses a preparation method, genes, application and a kit of the protein.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

A Lawsonia intracellularis flgE protein, wherein the amino acid sequence of the flgE protein is shown in SEQ ID No. 2.

Meanwhile, the invention also discloses a gene for encoding the L.intracellularis flgE protein, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.

Meanwhile, the invention also discloses application of flgE protein in preparing a detection kit.

In addition, the invention also discloses a kit, which comprises a pre-coating plate, wherein the protein coated in the pre-coating plate is flgE protein as described above.

The kit further comprises a sample diluent, an enzyme-labeled antibody, TMB substrate liquid, a stop solution, a concentrated washing solution, a sealing plate film, a diluent plate, a negative control liquid and a positive control liquid; the positive control solution is prepared from positive serum; the negative control solution is prepared from negative serum.

Finally, the invention also discloses a preparation method of the lawsonia intracellularis flgE protein, which is characterized in that the gene is cloned into a eukaryotic expression vector to construct a recombinant plasmid, and the recombinant plasmid is expressed by a prokaryotic cell to obtain the flgE protein.

In the above method, the method comprises the steps of:

Step 1: cloning a gene for encoding lawsonia intracellularis flgE protein into a prokaryotic expression vector pET-24a (+), and constructing a recombinant plasmid;

Step 2: transforming the recombinant plasmid into BL21 escherichia coli to obtain a recombinant expression strain;

Step 3: recombinant expression strain is induced by IPTG, cracked and centrifuged, and supernatant and sediment are taken; purifying by Ni-NTA affinity chromatography to obtain the recombinant truncated flgE protein of Lawsonia intracellularis.

Compared with the prior art, the invention has the following beneficial effects:

The invention constructs the coliform recombinant bacterium for expressing the lawsonia intracellularis truncated flgE protein based on the truncated lawsonia intracellularis flgE protein nucleotide sequence, and uses the recombinant bacterium for indirect ELISA detection of the lawsonia intracellularis flgE antibody, and the strain can efficiently express the lawsonia intracellularis flgE protein.

The truncated protein of the lawsonia intracellularis flgE provided by the invention adopts professional software to comprehensively analyze flgE genes, intercepts specific areas, is connected through a linker (GGSG), and has the advantages of specificity, accuracy, sensitivity and the like when being used for detecting flgE protein antibodies.

The invention further carries out codon optimization on the basis of the 287 amino acid sequence of the lawsonia intracellularis truncated flgE protein, and the optimized lawsonia intracellularis truncated flgE protein has the advantages of specificity, accuracy and sensitivity when used for detecting lawsonia intracellularis truncated flgE antibodies.

The invention further provides an indirect ELISA detection kit for the lawsonia intracellularis flgE protein antibody based on the optimized lawsonia intracellularis flgE protein. The kit has the advantages of strong specificity, high sensitivity, easy operation and long shelf life, and can be used for detecting lawsonia intracellularis antibody products and the like.

Drawings

FIG. 1 is a SDS-PAGE diagram of recombinant flgE protein expressed and identified in recombinant E.coli BL 21-flgE;

wherein M is a (14 kDa-140 kDa) protein Marker;1: the thalli are not induced by IPTG; 2: IPTG induced thalli; 3: crushing the supernatant; 4. and crushing the bacterial cells. The arrow indicates the position of the truncated recombinant protein;

FIG. 2 is a diagram showing the differences between the nucleotide sequence of truncated recombinant flgE protein of example 1 before and after genetic modification;

FIG. 3 SDS-PAGE of truncated recombinant flgE after purification;

Wherein M is a (14 kDa-140 kDa) protein Marker;1: eluent 1;2: eluent 2;3: eluent 3; 4. eluent 4. The arrow indicates the position of the truncated recombinant protein;

FIG.4 flgE truncated protein western blot results;

wherein M is Maker,1: results of actions of flgE truncated proteins with positive serum;

FIG. 5 flgE SDS-PAGE after whole gene protein purification;

Wherein M is a (14 kDa-140 kDa) protein Marker;1: flowing through liquid; 2: washing impurity liquid; 3: eluent (flgE whole gene target protein);

FIG. 6 flgE-1 SDS-PAGE after purification of truncated gene proteins;

Wherein M is a (14 kDa-140 kDa) protein Marker;1, other proteins; 2: flgE-1 truncating the protein of the gene;

FIG. 7 flgE-2 SDS-PAGE after purification of truncated gene proteins;

Wherein M is a (14 kDa-140 kDa) protein Marker;1: flgE-2 truncating the gene target protein;

FIG. 8 is a diagram showing comparison of protein sequences of comparative example 1, comparative example 2, comparative example 3 and example 1;

FIG. 9 flgE is a diagram of a secondary structural analysis of whole protein sequences.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

Before describing the examples and comparative examples, the sources of the relevant raw materials to which the invention relates are described:

LB medium: a biological organism: cargo number: b540111;

IPTG: isopropyl- β -D-thiogalactose: a biological organism: cargo number: b541007;

TritonX-100:4- (1, 3-tetramethylbutyl) phenyl-polyethylene glycol: aladine: cargo number: E-IR-R122;

imidazole: imidazole: microphone lin: cargo number: 288-32-4;

Ni-NTA affinity chromatography: his tag protein purification kit: holy life: cargo number: 20502ES10;

BCA kit: BCA protein concentration assay kit: shanghai Biyun biotechnology Co., ltd.): cargo number: P0012S;

PBST：PBS+5%Tween；

Skim milk powder-PBST blocking solution: a blocking solution for WB;

Sealing liquid: for ELISA blocking solution: 0.01M PBS+1% sodium caseinate+0.5% trehalose;

HPR-labeled goat anti-pig secondary antibody: purchased from Jackson corporation; cargo number 161245;

W-TMB color development liquid: a biological organism: c510025;

coating liquid: self-dispensing liquid; 0.05M carbonate buffer, ph=9.6;

Washing liquid: 1 XPBST;

Stop solution: 1mol/LHCL;

enzyme-labeled antibody: the working concentration of the goat anti-pig secondary antibody marked by HPR is diluted by an antibody protection solution in the company.

Example 1

Synthesis of Lawsonia intracellularis flgE Gene fragment and construction of prokaryotic expression plasmid flgE Gene fragment Synthesis

According to the gene sequence and amino acid sequence of flgE of (GenBank: NC_008011.1, refer to SEQ ID No. 3), the recombinant truncated gene sequence SEQ ID No.1 of Lawsonia intracellularis flgE protein, the amino acid sequence of which is SEQ ID No.2, was obtained by analyzing the target sequence for hydrophobicity and hydrophilicity, a transmembrane region and a signal peptide region. And carrying out codon optimization on the obtained gene sequence according to the compatibility of codons.

In order to more intuitively see the difference between SEQ ID No.2 and SEQ ID No.4, the results are shown in FIG. 8. It can be seen from the figure that SEQ ID No.2 differs from SEQ ID No.4 (full length) in that part of the sequence is removed from the middle and ending of the sequence. From the analysis of the secondary structure of the protein, the most antigenic and surface accessible parts of the sequence are strong, but the less antigenic and surface accessible parts of the sequence are also weak.

In the nucleotide sequence optimization (including GC analysis, CAI analysis, codon optimization) of example 1 of the present invention, the nucleotide sequence after optimization was submitted to the biological engineering (Shanghai) stock company, inc. of Biotechnology, suzhou, for sequence synthesis.

It is to be noted that the original nucleotide sequence of the L.intracellularis flgE protein gene is SEQ ID No.3; the original amino acid sequence of flgE protein is SEQ ID No.4;

the truncated recombinant flgE protein (SEQ ID No. 1) was prepared by delivering to the company limited by the biological engineering (Shanghai) company, the specific preparation method is:

Cloning flgE gene sequence (SEQ ID No. 2) into expression vector plasmid pET-24a (+) through restriction enzyme cleavage sites NdeI and XhoI, constructing recombinant plasmid pET-24a (+) -flgE, sequencing to obtain flgE gene sequence (SEQ ID No. 2) with correct sequencing result;

Transforming the recombinant plasmid pET-24a (+) -flgE into E.coliBL21 (DE 3) to obtain recombinant expression strain BL21-flgE, and transforming on a LB plate with kana resistance;

inducible expression and purification of recombinant flgE protein

Selecting recombinant expression strain pET-24a (+) -flgE monoclonal bacteria from a transformed flat plate, inoculating the monoclonal bacteria into 4mL of LB culture medium (the final concentration is 25ug/mL of kana), culturing until OD _600nm is 0.4-0.6, adding IPTG with the final concentration of 0.1mM into a test tube culture solution, inducing at 220rpm of a shaking table at 37 ℃ for 3-5, and increasing bacteria liquid which is not induced by the IPTG for comparison. The whole bacteria were boiled and identified, and the SDS-PAGE results are shown in FIG. 1.

The induced cells were collected by centrifugation and the cell pellet was resuspended in PBS at ph=7.2; bacteria were lysed by sonication for 30min until the bacterial solution became clear. Centrifuging again at 4deg.C and 8000rpm for 10min to obtain supernatant and thallus precipitate; the bacterial cell sediment is resuspended by PBS with the same volume as the supernatant to obtain sediment suspension;

The supernatant and the sediment suspension are respectively subjected to electrophoresis, a loading buffer solution is added and boiled for 10 min, and the supernatant is centrifugally taken at 4 ℃ for SDS-PAGE electrophoresis. And (3) performing stabilized electrophoresis by 90V, after the desert phenol blue indicator enters the separation gel, performing stabilized electrophoresis by 180V until the desert phenol blue band migrates to the bottom of the separation gel, taking out the gel, dyeing by using coomassie brilliant blue staining solution for 30min, and transferring the gel into a decolorizing solution until the background is clear. The results show that the lawsonia intracellularis recombinant truncated flgE protein is mainly present in the supernatant for soluble expression, and is then purified by a soluble purification method. SDS-PAGE results of soluble expression identification are shown in FIG. 1.

To more clearly illustrate the differences in flgE gene sequences of the present invention, the present invention provides the sequence of the flgE truncated recombinant gene in the lawsonia intracellularis sequence (GenBank: nc— 008011.1), with the amino acid sequence underlined being optimized (see fig. 2);

In order to more clearly illustrate the expression advantage of flgE gene sequences of the present invention after optimization, the present invention provides GC content analysis comparison results and codon fitness index CAI analysis comparison results.

The GC content of the original truncated recombinant flgE protein gene sequence is 44%; the GC content of the flgE protein gene sequence is 53%;

The codon adaptability index CAI of the original truncated recombinant flgE protein gene sequence is 0.67; the codon adaptability index CAI of flgE protein gene of the invention is 0.86.

Example 2

Large-scale expression, purification, antigenicity identification, large-scale expression and purification of L.intracellularis truncated recombinant flgE protein

Culturing recombinant expression strain BL21-flgE with positive expression in 2L of liquid LB culture medium added with kana, adding final concentration 0.1-mMIPTG when OD _600nm is 0.4-0.6, inducing at 37deg.C for 3-5 hr, and collecting thallus;

The induced cells were collected by centrifugation and the cell pellet was resuspended in PBS at ph=7.2; the ultrasonicator lyses the bacteria until the bacterial liquid becomes clear. Centrifuging again at 8000rpm for 10min to obtain supernatant and thallus precipitate; the supernatant was purified by Ni-NTA affinity chromatography.

After Ni-NTA affinity chromatography purification, eluent with different concentrations is obtained, and SDS-PAGE electrophoresis is carried out on the eluent. The result of electrophoresis is shown in FIG. 3.

The single eluted fractions were collected and dialyzed against.01M PBS, during which 3 changes were made. Protein concentration measurement is carried out by using a BCA kit, the protein concentration is 3mg/mL, the protein purity analysis reaches more than 95%, the concentration is diluted to 1mg/mL of working concentration, and the mixture is packaged and marked and stored at-80 ℃.

Westernblot identification of recombinant flgE protein of Lawsonia intracellularis

SDS-PAGE electrophoresis;

1) Transferring: after electrophoresis, placing the gel into a transfer membrane buffer solution for balancing for 10min; soaking PVDF membrane and filter paper with the same size as the glue in methanol for 10s, and then balancing in a transfer membrane buffer solution for 10min; placing a transfer film instrument in a horizontal position by adopting a semi-dry method, sequentially spreading a layer of filter paper, a PVDF film and an electrophoresis gel from bottom to top, placing a layer of filter paper, switching on a power supply, and regulating the voltage to 10V for transferring the film for 40min;

2) Washing: taking out the PVDF film after finishing film transfer, and washing the PVDF film 3 times by PBST for 5min each time;

3) Closing: placing PVDF membrane in (skimmed milk powder-PBST) sealing solution with mass fraction of 5% overnight, and washing in step 2;

4) Incubation resistance: PBST was used to 1: diluting lawsonia intracellularis positive serum according to a proportion of 100, incubating for 1h at room temperature, and washing in the same step 2;

5) Add PBST to 1:2000 proportion of diluted HPR marked goat anti-pig secondary antibody;

6) Incubating for 1h at 25 ℃, and washing in the same step 2;

7) Color development: soaking 10mL of the C solution of the W-TMB color development liquid kit for 10min, adding about 0.5mL of substrate solution A and 4 mu L of substrate solution B, fully and uniformly mixing, and reacting for 5-10min to generate blue product strips.

As shown in FIG. 4, the result of the imaging and development showed that the band of interest appeared at around 35 kDa.

Example 3

Preparation of ELISA kit for detecting lawsonia intracellularis flgE protein antibody

Determination of optimal antigen coating concentration, optimal serum dilution and dilution multiple of enzyme-labeled antibody

The L.intracellularis truncated recombinant flgE protein (prepared in example 2) was diluted with coating liquid doubling ratios to 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL and 0.25. Mu.g/mL ELISA plates per well and coated overnight at 4℃according to the checkerboard method; washing the washing solution for 3 times, beating to dry, adding the sealing solution, and incubating for 4 hours at 25 ℃; washing the washing liquid for 3 times, and removing the liquid and beating to dryness; adding a sealing liquid protection solution and incubating for 1h at 25 ℃; removing liquid and beating to dry; diluting Lawsonia intracellularis positive serum and Lawsonia intracellularis negative serum with sample dilutions at 1:50, 1:100, 1:200, and 1:400, and repeating each dilution once; adding 100 mu L of the mixture into each hole, reacting L h at 25 ℃, washing for 3 times, and beating to dryness; 100 μl of enzyme-labeled antibody was added to each well, and the dilution ratio was 1:20000, 1:40000, 1:80000, 1:160000, and the reaction was carried out at 25deg.C for 30min; washing for 3 times and beating to dry. Adding 100 mu L/hole of TMB substrate developing solution, carrying out light-proof reaction at room temperature of 25 ℃ for 15min, adding 50 mu L/hole of stop solution, and measuring OD _450nm value by using an enzyme-labeling instrument, wherein the measurement results are shown in Table 1:

TABLE 1P/N Range of optimal coating concentration of antigen, optimal dilution of serum and dilution of enzyme-labeled antibody

Antigen coating concentration	8μg/mL	4μg/mL	2μg/mL	1μg/mL
					P/N range	2.25-4.04	2.16-4.30	2.58-4.10	3.6-4.5
Serum dilution factor	50 Times dilution	100-Fold dilution	200 Times dilution	400 Times dilution
					P/N range	2.30-2.62	3.61-4.11	2.51-3.32	2.52-3.82
Dilution of enzyme-labeled antibody	2W	4W	8W	16W
					P/N range	1.91-2.25	3.54-4.52	2.54-3.65	2.62-3.3.21

By calculating the relative OD _450nm value of positive control serum and the relative OD _450nm value (P/N value) of negative control serum and comparing the ranges of the P/N values under different conditions, the antigen coating concentration with relatively larger P/N value is determined to be 1 mug/mL, the optimal dilution of serum is 1:100, and the dilution multiple of enzyme-labeled secondary antibody is 40000 as the optimal reaction condition of ELISA.

Determination of sample incubation time

Based on the optimal experimental conditions of the measurement, sample incubation time is screened, after 1:100 times of dilution is carried out on each hole of clinical samples, the samples react for 30min, 60min, 90min and 120min at room temperature respectively, ELISA tests are carried out on 2 negative serum and 2 positive serum, OD _450nm values of each hole are measured, and the measurement results are shown in Table 2:

Table 2 selection of serum incubation time

By calculation, the optimal reaction time for incubation of the sample was calculated as 30min, where the ratio (P/N) of the average OD _450nm value of the positive sample to the average OD _450nm value of the negative sample was relatively large.

Determination of enzyme-labeled antibody reaction time

Based on the optimal experimental conditions, TMB substrate color development time is screened, 100 mu LTMB substrate liquid is added into each hole, the reaction is carried out for 30min, 60min and 90min at room temperature in a dark way, ELISA tests are carried out on 2 parts of negative serum and 2 parts of positive serum, OD _450nm values of each hole are measured, and the measurement results are shown in Table 3:

Table 3 selection of enzyme-labeled antibody incubation times

By calculation, the optimal reaction time of the enzyme-labeled antibody is calculated as 30 minutes with a relatively large ratio (P/N) of the average OD _450nm value of the positive sample to the average OD _450nm value of the negative sample.

Determination of TMB substrate development time

On the basis of the optimal experimental conditions, TMB substrate color development time is screened, 100 mu LTMB substrate liquid is added into each hole, the reaction is carried out at room temperature and in a dark place for 5min, 10min, 15min and 20min respectively, ELISA tests are carried out on 2 parts of negative serum and 2 parts of positive serum, OD _450nm values of each hole are measured, and the measurement results are shown in Table 4:

TABLE 4 color development time comparison

By calculation, 15min, which is a relatively large ratio (P/N) of the average OD _450nm value of the positive sample to the average OD _450nm value of the negative sample, was used as the optimal color development time of the TMB substrate.

Determination of ELISA cut off

Diluting 117 normal pig serum samples (Endocon antibody detection negative samples) according to 1:00, performing indirect ELISA under the optimal condition of screening, setting a negative and positive control hole, performing statistical analysis on the detection result to obtain OD _450nm, calculating an average value (X) and a Standard Deviation (SD) of an S/P value, and judging that the sample is positive when the S/P value is X+3SD (namely, the S/P value is 0.6); S/P is X+2SD, that is, S/P value is 0.4), the result is negative, and the result is suspicious between the two.

Example 4

Assembling a lawsonia intracellularis recombinant flgE protein antibody detection kit:

Diluting the lawsonia intracellularis recombinant flgE protein to 1 mug/mL with a coating solution, coating an ELISA plate, adding 100 mug/hole, standing overnight at 4 ℃, washing 3 times with a washing solution, and then beating to dry; adding a sealing solution, adding 200 mu L of sealing solution into each hole, and incubating for 4 hours at 25 ℃; then washing 3 times with washing liquid, beating and drying, adding protective liquid, adding 200 mu L of protective liquid into each hole, incubating for 1h at 25 ℃, discarding liquid, beating and drying; sealing the sealing plate film to obtain a pre-coated plate of lawsonia intracellularis flgE protein, and storing at 4 ℃ for later use;

And (3) assembling a pre-coated plate of the lawsonia intracellularis recombinant flgE protein, negative control serum, positive control serum, sample diluent, enzyme-labeled antibody, substrate chromogenic solution TMB, stop solution and concentrated washing solution to obtain the lawsonia intracellularis flgE protein antibody indirect ELISA detection kit.

Adding diluted sample to be detected, negative control serum (lawsonia intracellularis negative serum) and positive control serum (lawsonia intracellularis positive serum containing flgE protein antibodies) into a coating plate, wherein each hole is 100 mu L, 1 hole is arranged in the sample to be detected, 2 holes are respectively arranged in the negative control serum and the positive control serum, and incubating at 25 ℃ for 30min; washing 3 times by using a washing liquid; then adding enzyme-labeled antibody, 100 mu L of each hole, and incubating at 25 ℃ for 30min; washing 3 times by using the washing liquid, and finally, beating the washing liquid on the absorbent paper; adding 100 mu L of substrate color development liquid into each hole, and developing for 15min at room temperature and 25 ℃ in a dark place; finally, 50 mu L of stop solution is added into each hole, and an enzyme-labeled instrument is used for measuring an OD _450nm light absorption (OD _450nm) value within 15min;

And (3) result judgment: the test is satisfied under the condition that the average value of the positive control hole OD _450nm is required to be larger than 1, and the average value of the negative control hole OD _450nm is required to be smaller than 0.3; the S/P of the sample is more than or equal to 0.6, and the sample is judged to be positive; and the sample S/P is less than or equal to 0.4, and the sample is judged as negative. When 0.4< S/P <0.6 is suspicious, the resampling detection needs to be continuously tracked.

Example 5

Evaluation of the compliance (sensitivity, specificity) Effect of the Indirect ELISA detection of the recombinant flgE protein antibody of Lawsonia intracellularis

154 Samples of Lawsonia intracellularis positive serum and 67 samples of Lawsonia intracellularis negative positive serum, each of which includes two samples of African swine fever positive serum, foot-and-mouth disease type-O positive serum, foot-and-mouth disease type-A positive serum, swine fever positive serum and pseudorabies positive serum, each of which was positive by using the Haemophilus antibody, were tested according to the method of example 4, and the test results are shown in Table 5:

Table 5: clinical serum compliance test of research and development kit

The detection results are as follows: 134 parts of the research and development kit can be detected by 144 parts of lawsonia intracellularis positive serum; 57 parts of the research and development kit can be detected by 62 parts of lawsonia intracellularis negative serum; sensitivity was 93.06% and specificity 91.94%; the overall compliance was 92.72%.

Example 6

High-temperature stability experiment of lawsonia intracellularis flgE antibody detection kit

3 Batches of Lawsonia intracellularis flgE antibody detection kit were prepared and placed in a 37℃incubator for 15 days of thermal stability. Meanwhile, the positive control of day 7 and day 15 was performed during the period compared with the kit stored at 4 ℃, and the degradation rate of the OD _450nm value of the clinical sample is shown in table 6.

Table 6: kit overall stability detection

A thermal stability experiment at 37 ℃ was performed by preparing 3 batches of L.intracellularis recombinant flgE protein antibody indirect ELISA detection kit. The degradation rate of the OD _450nm value of the positive control and the clinical sample is lower than 20% by the stability detection on the 4 th day and the 15 th day, and the quality guarantee period of the detection kit can be stored for more than 14 months under the condition of 4 ℃ according to a thermal stability experiment.

Comparative example 1

L.intracellularis flgE genome ((GenBank: NC-008011.1, reference SEQ ID No. 3) Gene synthesis, protein expression.

The secondary structure of the protein was analyzed if shown in FIG. 9. The results show that: the alpha-helix occupies a small amount and is mainly positioned in the sections 40-48, 81-91, 293-300, 453-463, 498-519 and 679-690, and the lengths are shorter. The β -sheet is densely and uniformly distributed among the sequences, predominantly at the 1-14、19-49、54-73、103-116、121-136、166-187、194-209、223-255、264-273、280-293、296-309、314-321、326-338、351-358、372-381、388-402、432-444、449-462、479-507、526-531、535-543、546-556、570-605、631-637、651-668、673-691.β- corners, predicted to be uniformly dispersed in the sequences by the Gamier-Robson method. The random coil is located primarily in sections 14-18, 73-82, 134-141, 148-153, 159-163, 213-221, 259-264, 414-421, 519-525, 628-632. The flexible region is mainly located in 50-63、73-85、96-123、132-155、158-166、186-204、210-226、251-266、247-282、306-317、322-334、345-349、382-397、407-432、443-454、475-494、531-539、543-551、557-570、608-657、664-679 sections and is uniformly distributed in the amino acid sequence, which shows that the protein has good flexibility. The antigenic regions are predominantly located in the 15-23、35-54、74-122、133-169、185-199、208-228、234-244、251-266、274-297、307-318、321-334、343-351、356-373、382-395、403-434、443-452、474-497、508-525、529-571、605-636、643-656、664-677 region, suggesting that these regions have a greater chance of becoming dominant epitopes. The surface accessibility regions are mainly distributed in the 79-85、98-111、135-141、187-200、211-236、252-259、274-282、291-297、323-330、408-420、425-434、475-501、514-521、531-538、544-549、607-617、622-630、 region, suggesting that these regions may be located on the molecular surface of flgE protein, with a high probability of being epitopes.

The flgE gene of comparative example 1 was synthesized by the company division of bioengineering (Shanghai);

2. The sequence of interest was added with cleavage sites, recombinant vectors, expression and purification methods as in example 1; the purification result of the target protein is shown in figure 5;

3. Kit preparation the same as in example 3 and example 4, corresponding clinical sample detection systems were established;

4. effect evaluation the results of the test are shown in table 7 as in example 5:

Table 7: clinical serum compliance test of research and development kit

The detection results are as follows: 128 parts of the lawsonia intracellularis positive serum can be detected by the research and development kit; 50 parts of the research and development kit can be detected by 62 parts of lawsonia intracellularis negative serum; sensitivity was 88.89% and specificity 80.65%; the overall compliance was 86.41%.

Comparative example 2

A truncated gene sequence of flgE (SEQ ID No. 5) was also optimized from the flgE whole gene (GenBank: NC-008011.1, see SEQ ID No. 3) at the same time as the truncated gene sequence of L.intracellularis flgE (SEQ ID No. 1). The resulting sequence was codon optimized according to codon compatibility. The protein sequence expressed by the gene sequence is 6 (SEQ ID No. 6). In order to more intuitively see the difference between SEQ ID No.6 and SEQ ID No.4, the results are shown in FIG. 8. It can be seen from the figure that the difference between SEQ ID No.6 and SEQ ID No.4 (full length) is that part of the sequence is removed in the middle of the ending with the sequence. From the analysis of the secondary structure of the protein, the more antigenic part was retained, the less antigenic part was removed, and the cleavage region was noted to avoid the beta sheet.

The flgE gene of comparative example 2 was synthesized by the company division of bioengineering (Shanghai);

2. the sequence of interest was added with cleavage sites, recombinant vectors, expression and purification methods as in example 1; the purification result of the target protein is shown in FIG. 6;

3. Kit preparation the same as in example 3 and example 4, corresponding clinical sample detection systems were established;

4. Effect evaluation the same as in example 5, test results are shown in table 8:

table 8: clinical serum compliance test of research and development kit

The detection results are as follows: 129 parts of lawsonia intracellularis positive serum can be detected by the research and development kit; 54 parts of the research and development kit can be detected by 62 parts of lawsonia intracellularis negative serum; sensitivity was 89.58% and specificity was 87.10%; the overall compliance was 88.83%.

Comparative example 3

The truncated gene sequence of flgE (SEQ ID No. 6) was also optimized from the flgE whole gene (GenBank: NC-008011.1, ref. SEQ ID No. 3) at the same time as the truncated gene sequence of L.intracellularis flgE (SEQ ID No. 1) was obtained. The protein sequence expressed by the gene sequence is 8 (SEQ ID No. 8). The resulting sequence was codon optimized according to codon compatibility. The protein sequence expressed by the gene sequence is 8 (SEQ ID No. 8). In order to more intuitively see the difference between SEQ ID No.8 and the original sequence, the results are shown in FIG. 8. From the figure, it can be seen that SEQ ID No.8 is shorter in sequence than SEQ ID No.4 (full length), and from the analysis of the secondary structure of the protein, the sequence has both surface accessibility while taking antigenicity into consideration, and the part of both sequences is reserved.

The flgE gene of comparative example 3 was synthesized by the company division of bioengineering (Shanghai);

2. the sequence of interest was added with cleavage sites, recombinant vectors, expression and purification methods as in example 1; the purification result of the target protein is shown in FIG. 7;

3. Kit preparation the same as in example 3 and example 4, corresponding clinical sample detection systems were established;

4. effect evaluation the same as in example 5, test results are shown in table 9:

Table 9: clinical serum compliance test of research and development kit

The detection results are as follows: 130 parts of the research and development kit can be detected by 144 parts of lawsonia intracellularis positive serum; 55 parts of the research and development kit can be detected by 62 parts of lawsonia intracellularis negative serum; sensitivity was 90.28% and specificity was 88.71%; the overall compliance was 89.81%.

By comparison, it can be seen that the truncated recombinant flgE protein of the present invention has the following advantages:

A large number of sequence analyses are carried out on flgE protein sequences, and target sequences with good antigenicity and hydrophilicity are selected for truncated expression according to hydrophilicity and antigenicity prediction carried out on the target sequences, so that the target sequences have good antigenicity and hydrophilicity.

After determining the truncated recombinant flgE sequence, the invention optimizes the codon according to the codon preference, and under the condition of changing the amino acid sequence, the optimal set of codon system of the expression host is used, so that the success rate of the expression of the truncated recombinant flgE is greatly improved.

The invention adopts truncated flgE sequence to reduce original target gene sequence from 2073bp to 861bp, and reduce gene synthesis cost.

The sensitivity and the specificity of the kit prepared by adopting the truncated flgE protein are higher than those of the kit prepared by the background technology.

Meanwhile, the comparison of the comparative example 1 with the comparative examples 2, 3 and the examples shows that the effect of the full-length flgE protein is not as good as that of the truncated protein.

By comparison with examples 2 and 3, both of them 3 had both antigenicity and surface accessibility at the time of selecting sequence fragments. But the antigenic and accessibility ratios are different, which affects the properties of the protein. Comparative example 2 has the highest antigenic duty cycle, comparative example 3 has the highest surface accessibility duty cycle, and examples have the highest antigenic and surface accessibility duty cycle in the middle, and also have fragments with lower partial antigenicity and lower surface accessibility. This indicates that the merits of the proteins used in the kit are related to at least three factors:

1. the antigenic high and low parts in the sequence are selected.

2. The surface accessibility is selected from the sequence.

3. Selection of beta sheet in the sequence.

The three have close relativity.

The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to the above process steps, which do not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.

Claims (7)

1. The lawsonia intracellularis flgE protein is characterized in that the amino acid sequence of the flgE protein is shown as SEQ ID No. 2.

2. A gene encoding the lawsonia intracellularis flgE protein according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 1.

3. Use of the flgE protein of claim 1 for the preparation of a test kit.

4. A kit comprising a pre-coated plate wherein the coated protein is the flgE protein of claim 1.

5. The kit of claim 4, further comprising a sample diluent, an enzyme-labeled antibody, a TMB substrate solution, a stop solution, a concentrated washing solution, a sealing plate membrane, a dilution plate, a negative control solution, a positive control solution; the positive control solution is prepared from positive serum; the negative control solution is prepared from negative serum.

6. A method for preparing the lawsonia intracellularis flgE protein according to claim 1, wherein the gene according to claim 2 is cloned into a eukaryotic expression vector to construct a recombinant plasmid, and the recombinant plasmid is expressed by prokaryotic cells to obtain flgE protein.

7. The method according to claim 6, comprising the steps of:

Step 1: cloning a gene for encoding lawsonia intracellularis flgE protein into a prokaryotic expression vector pET-24a (+), and constructing a recombinant plasmid;

Step 2: transforming the recombinant plasmid into BL21 escherichia coli to obtain a recombinant expression strain;

Step 3: recombinant expression strain is induced by IPTG, cracked and centrifuged, and supernatant and sediment are taken; purifying by Ni-NTA affinity chromatography to obtain the recombinant truncated flgE protein of Lawsonia intracellularis.