CN108484766B - Anti-toxoplasma Thioredoxin nano antibody and coding gene and application thereof - Google Patents

Abstract

The invention discloses a nano antibody for resisting toxoplasma Thioredoxin and a coding gene and application thereof, wherein the VHH chain amino acid sequence of the nano antibody is shown as SEQ ID No. 4. The nanobody comprises two VHH chains. The anti-toxoplasma TRX nano antibody is prepared by immunizing camel with toxoplasma TRX antigen to obtain an anti-TRX nano antibody library, and then screening the anti-toxoplasma TRX nano antibody library to obtain the anti-TRX nano antibody with better performance, has high water solubility and conformation stability, stronger antigen affinity and excellent tissue penetration capacity, can efficiently detect toxoplasma, and can be used for preparing a toxoplasma detection kit.

Description

Anti-toxoplasma Thioredoxin nano antibody and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a toxoplasma Thioredoxin (TRX) resistant nano antibody, and a coding gene and application thereof.

Background

Toxoplasmosis is a zoonotic protozoal disease caused by Toxoplasma gondii (t.gondii). Toxoplasma gondii is transmitted to fetus through placenta mainly in the acute infection stage of mother, once it invades placenta, 95% of fetus can not be survived, and can directly affect fetus development, severe teratogenesis and even death, and can make pregnant woman miscarriage, dead birth and premature delivery, and increase pregnancy complication. The congenital toxoplasmosis patients have typical clinical manifestations, and generally have four characteristics of hydrocephalus or cerebellar malformation, brain calcific focus, chorioretinitis, mental dyskinesia such as mental retardation, spasm, paralysis and the like. The fetal infection rates were 17%, 25% and 65% in the early, middle and late gestation, respectively, and fetal damage was most severe when infection occurred at weeks 8-24. With the change of modern dietary habits and the increase of domestic pets, the infection rate of toxoplasmosis in China is on a rising trend year by year. The 2006 + 2012 survey data shows: the infection rate of normal healthy people in three cities of Shanghai, Changchun and Daqing exceeds 11 percent, so that the total number of infected people is estimated to be about 1.4 hundred million. In countries such as france, usa and Poland with higher infection rate, there is mandatory monitoring regulation for women of childbearing age and pregnant women, and better effect of preventing congenital toxoplasmosis is achieved. In China, the infection detection of toxoplasma is also listed as one of the five indicators of the eugenic (TORCH) of pregnant women.

Clinical diagnosis case judgment of toxoplasmosis requires combination of clinical symptoms and epidemic history, and simultaneously conforms to any one of etiological isolation positive, nucleic acid positive or Circulating antigen (CAg) positive. The traditional etiology examination methods such as direct microscopic examination and animal inoculation are time-consuming and labor-consuming, have low detection rate and are easy to miss detection. The PCR and loop-mediated isothermal amplification nucleic acid rapid detection technology established by taking SAG1, B1 and 529bp repeat sequences and the like in a toxoplasma genome as target genes has the advantage of high sensitivity, has important significance for early diagnosis of toxoplasma infection, but aerosol pollution is easily formed by the molecular diagnosis technology, and certain false positive exists in most laboratories in China at present because the laboratories cannot be strictly partitioned. The detection of the circulating antigen can reflect the infection of the insect and lotus, and is an index for early and present infection and curative effect assessment. However, the toxoplasmosis detection reagents currently approved by the national food and drug administration only contain the detection of IgG and IgM antibodies. The antibody level measurement alone cannot fully reflect the toxoplasma infection condition, and is very likely to cause missed diagnosis of a part of patients with the existing diseases. The positive CAg can be used as the reference basis for acute and present infection; positive IgM antibody has early diagnosis value; IgG antibody positive can be diagnosed as previous infection, and the triple combined detection reagent can achieve the purpose of judging the course of disease, however, because the content of CAg in an individual is low, the content of CAg is rapidly reduced to a low level particularly after 2 weeks of infection. For mild and chronic patients, the detection rate of the conventional CAg detection method is low, and the requirement cannot be met.

TRX is a widely distributed multifunctional protein, which is a very effective antioxidant substance, and the redox activity of the TRX plays a plurality of functions in vivo, such as antioxidation, regulation of cell proliferation, differentiation and apoptosis, active oxygen free radical removal, tissue protection, and is related to cell apoptosis, immune regulation, embryonic growth and development and the like; in addition, TRX can effectively reduce active oxygen generated by host immune cells caused by insect infection and reduce damage caused by metabolites, thereby being beneficial to the long-term survival of the insects in vivo. Research shows that TRX has better immune protection effect and diagnostic value in some parasites.

A natural light chain-deleted functional Heavy chain antibody (HCAb) exists in a camel body, and the variable region of the Heavy chain antibody is cloned to obtain a minimum antigen-binding fragment, namely a Nanobody (Nb). The great advantage of nanobodies is their small size. The molecular weight is only 15KDa, which is less than one tenth of that of the traditional antibody (160 KDa). Compared with a common antibody, Nb also has the advantages of high water solubility and conformation stability, stronger antigen affinity, excellent tissue penetration capacity, easy in-vitro expression, humanized modification and the like, and the characteristics of Nb enable the Nb to show wide application prospects in the fields of diagnosis, detection and treatment.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a nano antibody for resisting toxoplasma gondii TRX, a coding gene and application thereof.

A VHH chain of an anti-Toxoplasma gondii Thioredoxin nano antibody comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID No.1, the amino acid sequence of CDR2 is shown in SEQ ID No.2, and the amino acid sequence of CDR3 is shown in SEQ ID No. 3.

The amino acid sequence of the VHH chain is shown in SEQ ID No. 4.

The invention also provides a nano antibody for resisting the toxoplasma gondii Thioredoxin, which comprises two VHH chains.

The invention also provides genes encoding the VHH chains, or the nanobodies.

The nucleotide sequence of the gene is shown in SEQ ID No. 5.

The invention also provides a recombinant expression vector containing the gene.

The invention also provides a gene engineering cell which is obtained by introducing the recombinant expression vector into a host cell.

The host cell is Escherichia coli, yeast or CHO cell.

The invention also provides application of the nano antibody in preparation of a toxoplasma gondii detection kit.

The anti-toxoplasma TRX nano antibody is prepared by immunizing camel with toxoplasma TRX antigen to obtain an anti-TRX nano antibody library, and then screening the anti-toxoplasma TRX nano antibody library to obtain the anti-TRX nano antibody with better performance, has high water solubility and conformation stability, stronger antigen affinity and excellent tissue penetration capacity, can efficiently detect toxoplasma, and can be used for preparing a toxoplasma detection kit.

Drawings

FIG. 1 is a SDS-PAGE electrophoresis detection result chart of Toxoplasma gondii TRX protein after recombinant expression and purification.

FIG. 2 is a diagram showing the results of PCR detection of colonies, wherein lane M is a standard DNA Marker D L2000, and lanes 1 to 18 represent 18 colonies, respectively.

FIG. 3 is a diagram showing the results of the detection of 9 anti-TRX nanobodies E L ISA.

FIG. 4 is a graph showing the results of the evolutionary tree analysis of the amino acid sequences of 9 anti-TRX nanobodies.

FIG. 5 is the plasmid map of prokaryotic expression recombinant vector of clone No. 37 anti-TRX nano antibody.

FIG. 6 is a diagram showing the results of prokaryotic expression and purification electrophoresis detection of clone No. 37 anti-TRX nano antibody.

Detailed Description

Example 1

Recombinant TRX protein, the steps are as follows:

(1) extracting Toxoplasma gondii cDNA by Trizol method, designing PCR amplification primer according to Toxoplasma gondii TRX gene sequence (GenBank number: XM-002370147) on NCBI, and performing PCR amplification to obtain TRX gene segment;

(2) constructing a TRX gene segment into a pET-28a expression vector, and transforming competent cells B L21;

(3) IPTG induction expression, thallus collection and ultrasonic crushing, wherein the protein is mainly expressed in supernatant, and the recombinant protein 15KDa is consistent with the expected size; the supernatant was purified by nickel column and dialyzed for renaturation, and the results of SDS-PAGE are shown in FIG. 1.

Example 2

Constructing a toxoplasma specificity nano antibody library, comprising the following steps:

(1) purifying to obtain toxoplasma gondii recombinant TRX protein, mixing 1mg of toxoplasma gondii recombinant TRX protein with Freund adjuvant in equal volume, immunizing a camel (Camelus bactrianus), performing experiments in inner Mongolia red peak for 2015 5 months, immunizing for 3 times once a week, and stimulating B cells to express antigen-specific nano antibody;

(2) after 3 times of immunization, 100ml of camel peripheral blood lymphocytes are extracted and total RNA is extracted;

(3) synthesizing cDNA through reverse transcription, and amplifying to obtain a heavy chain variable region gene VHH by utilizing nested PCR (nest PCR);

(4) carrying out double enzyme digestion on amplification products of Nest PCR and a phagemid vector pHEN 4 by using restriction endonuclease, connecting purified enzyme digestion products according to the mol ratio of optimized VHH insert fragments to vector molecules of 3: 1, carrying out electrotransformation in competent cells TG1, constructing a Toxoplasma gondii TRX nano antibody library, determining the library capacity, and measuring the size of the constructed library capacity (living bacteria number) to be 3.6 × 10 by plate counting⁹。

TABLE 1

Positive rate	Library capacity	Titer (pfu/ml)
			100％	3.9×109	1.83×1013

Colony PCR results are shown in FIG. 2, a band of 500-750bp is amplified from 18 single colonies in the experimental group, and the positive rate is 100%, so that the actual library volume of the initial library is 3.9 × 10⁹The titer of the constructed nanobody library was 1.83 × 10 by plaque counting using a double-layer agar plate method¹³pfu/ml。

Example 3

And (4) panning of specific nano antibodies. The method comprises the following steps:

(1) the solution was dissolved in 100mM NaHCO₃Coating 20 mu g of toxoplasma gondii recombinant TRX antigen with pH of 8.2 on a NUNC enzyme label plate, and standing overnight at 4 ℃;

(2) adding 100 mul of milk with the mass concentration of 3% in the next day, and sealing for 2h at room temperature;

(3) after 2h, 100. mu.l 2 × 10 was added¹¹tfu contains helper phage of the above nanometer antibody library, and acts for 1h at room temperature;

(4) washing 10 times with 0.05% PBS + Tween-20 in the first panning/20-25 times in the second panning to remove the non-specifically bound phage;

(5) phages specifically bound to toxoplasma recombinant TRX antigen were dissociated with 100mM TEA (triethylamine) and infected with escherichia coli TG1 in logarithmic growth phase, cultured at 37 ℃ for 1h, and phages were generated and purified for the next round of panning, gradually enriched, and the results are shown in table 2.

TABLE 2

Number of panning rounds	TRX concentration (μ g/m L)	Starting quantity (cfu)	Elutriation quantity (cfu)	Degree of enrichment
					1	300	2.80×1011	1.06×106	2.64×105
2	60	3.11×1011	5.04×107	6.17×103
					3	20	2.84×1011	6.14×108	4.63×102

Example 4

Screening of specific single positive clones by phage enzyme-linked immunosorbent assay (E L ISA) was performed as follows:

(1) from the phage-containing cell culture dishes after the panning described above, 96 individual colonies were picked and inoculated into TB medium containing 100. mu.g/ml ampicillin, and after growth to logarithmic phase, they were cultured overnight at 28 ℃ with the addition of IPTG at a final concentration of 1 mM.

(2) Obtaining crude antibody by using an osmosis method, transferring the antibody into an E L ISA plate coated by antigen, and standing for 1 hour at room temperature;

(3) unbound antibody was washed away with PBST, mouse anti-His antibody (mouse anti-His tag antibody, R & Dsystem) was added, and left at room temperature for 1 hour;

(4) unbound antibody was washed away with PBST and anti-mouse alkaline phosphataseconjugate (goat anti-mouse AP-labeled antibody, sigma) was added.

(5) Unbound antibody was washed away with PBST, alkaline phosphatase developing solution was added, and absorbance was read at 450nm on a microplate reader.

(6) And when the OD value of the sample well is more than 2.1 times of that of the control well, judging the sample well to be a positive cloning well.

(7) The positive clone well was transferred to TB medium containing 100. mu.g/ml ampicillin, and the plasmid was extracted and sequenced.

Among them, 9 clones with higher absorbance were shown in FIG. 3.

From the sequencing results, Vector was applied

11.5(Invitrogen, USA) and

the software analyzed individual clones, and identified strains with identical CDR1, CDR2, and CDR3 sequences as identical clones, while sequences that differ as different clones.

The gene sequence of the anti-TRX positive clone is translated into an amino acid sequence, the amino acid sequences are compared, the gene sequence and the amino acid sequence of the positive clone are compared and analyzed by an evolutionary tree, and the difference in the amino acid composition of the key site between the positive clones is determined, thereby preliminarily determining the epitope heterology aimed by the positive clones (fig. 4).

TABLE 3

Cloning	Coating TRX	Without being coated
			TRX-3	1.318	0.113
TRX-6	1.315	0.112
			TRX-9	1.079	0.109
TRX-12	0.917	0.101
			TRX-13	1.242	0.101
TRX-14	1.663	0.102
			TRX-24	1.080	0.096
TRX-28	0.911	0.086
			TRX-37	1.674	0.093

According to results of phage E L ISA, clone No.3, 6, 9, 12, 13, 14, 24, 28, 37 of 40 randomly selected positive clones has better antigen recognition, wherein clone No. 37 is the best, data are shown in Table 3, the gene sequence of the anti-TRX nanobody of clone No. 37 is shown in SEQ ID No.5, the amino acid sequence of VHH chain of nanobody is shown in SEQ ID No.4, each nanobody molecule has two VHH chains, the amino acid sequence of VHH chain is composed of 4 framework regions FR and 3 complementarity determining region CDRs, the framework regions FR comprises FR1 shown in SEQ ID No.6, FR2 shown in SEQ ID No.7, FR3 shown in SEQ ID No.8 and FR4 shown in SEQ ID No.9, the complementarity determining region CDRs comprise CDR1 shown in SEQ ID No.1, CDR2 shown in SEQ ID No.2 and CDR3 shown in SEQ ID No. 3.

Example 5

Prokaryotic expression of anti-toxoplasma gondii TRX nano antibody (clone No. 37) comprises the following steps:

1. cloning

(1) Primer design

Primers were designed based on the obtained gene sequence of the nanobody, and the specific sequences of the primers are shown in Table 4.

TABLE 4

Primer and method for producing the same	Sequence (5 '-3')
		VHH-F	CCCaagcttATGAAATACCTATTGCCTACGGC (HindIII restriction site underlined)
VHH-R	ATTTgcggccgcATGATGATGATGATGGTGCAGGT (Not I restriction site underlined)

(2) Amplification of

Using the obtained positive clone (No. 37 clone) of the anti-toxoplasma TRX nano antibody as a template, amplifying by using VHH-F and VHH-R primers to obtain a target fragment of the nano antibody, wherein the target fragment is a 500bp gene sequence, and the reaction procedure is as follows: 5min at 95 ℃; 95 ℃ for 50s, 64 ℃ for 45s and 72 ℃ for 1 min; 10min at 72 ℃. The target fragment is purified and recovered by a gel recovery kit, and the cloned vector pMD19T-simple is connected and then sent to a sequencing company for sequencing.

(3) The target fragment (pMD 19T-single plasmid with the fragment with correct sequencing) and pET32a prokaryotic expression vector are cut by double restriction, and the target fragment and the linearized pET32a vector are connected to construct a VHH-pET32a recombinant vector (figure 5).

2. Transformation of

(1) Dissolving competent cell B L21 preserved at-70 deg.C on ice, adding recombinant vector in clean bench, mixing, and ice-cooling for 25 min;

(2) rapidly carrying out ice bath for 2min after pre-heating to 42 ℃ in a water bath kettle for 45 s;

(3) adding 1ml of non-resistant L B liquid culture medium into an ultra-clean workbench, and performing shake culture on a shaking table at the temperature of 37 ℃ for 1 h;

(4) centrifuging at 5000rpm for 5min, discarding supernatant, blowing suspension liquid, spreading on L B plate with Amp resistance, and culturing at 37 deg.C overnight;

3. culture expression

(1) Positive colonies with correct sequencing were grown up and inoculated at a concentration of 1: 100 into 200ml containing Amp⁺The liquid L B medium is cultured by shaking on a shaker at 37 ℃;

(2) when the bacterial suspension was cultured to OD600 of 0.6, 1ml of the bacterial suspension was taken out and labeled for 0 h. Adding 100mM IPTG into 200ml culture solution according to the proportion of 1: 100, and continuing culturing;

(3) taking out 1ml of the 200ml bacterial liquid every 2 hours, continuously taking 4 times, and sequentially marking as 2, 4, 6 and 8;

(4) centrifuging at 12000rpm for 1min, discarding supernatant, adding 15 μ l 1 × SDS buffer, blowing the sample into uniform boiling water bath for 10min, ice-cooling for 2min, centrifuging at 12000rpm for 5min, and storing at-20 deg.C.

4. Expression detection

(1) Preparing SDS-PAGE electrophoresis gel with 12% separation gel and 5% concentration gel;

(2) adding an electrophoresis buffer solution of 1 × into an electrophoresis tank, putting the prepared gel into the electrophoresis tank, adding a sample and a protein marker, adjusting the initial voltage to be 80V when the upper layer concentrated gel presses the sample to a straight line, adjusting the voltage to be 120V, and stopping electrophoresis when the bromophenol blue dye migrates to the bottom of the gel;

(3) taking out the gel, placing the gel in a glass plate, adding a Coomassie brilliant blue staining solution which is submerged in the gel, and staining for 1-2 h on a shaking table with the rotating speed of 70 rpm;

(4) discarding the Coomassie brilliant blue staining solution, adding a decolorizing solution, and decolorizing on a shaking table;

(5) and (4) placing the decolored protein gel under a gel imager to observe the protein expression condition, and taking pictures.

As shown in FIG. 6, lane 1 shows the result before purification, lane 2 shows the result after purification, and the arrow indicates that the target protein is expressed.

Sequence listing

<110> Zhejiang province academy of medical science

<120> anti-toxoplasma Thioredoxin nano antibody and coding gene and application thereof

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Claims (9)

1. A VHH chain of a nano antibody of anti-Toxoplasma gondii TRX comprises three complementarity determining regions CDR1, CDR2 and CDR3, and is characterized in that the amino acid sequence of CDR1 is shown as SEQ ID No.1, the amino acid sequence of CDR2 is shown as SEQ ID No.2, and the amino acid sequence of CDR3 is shown as SEQ ID No. 3.

2. The VHH chain according to claim 1, characterized in that the amino acid sequence is according to SEQ ID No. 4.

3. A nanobody against toxoplasma TRX comprising two VHH chains according to claim 2.

4. A gene encoding a VHH chain according to claim 1 or 2 or a nanobody according to claim 3.

5. The gene of claim 4, wherein the nucleotide sequence is as shown in SEQ ID No. 5.

6. A recombinant expression vector comprising the gene of claim 5.

7. A genetically engineered cell obtained by introducing the recombinant expression vector of claim 6 into a host cell.

8. The genetically engineered cell of claim 7, wherein the host cell is an E.coli, yeast, or CHO cell.

9. Use of the nanobody of claim 3 in the preparation of toxoplasma gondii detection kits.

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