CN104650224B - It is a kind of for SEA nano antibody and its coded sequence and application - Google Patents
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Abstract
The invention discloses be directed to soluble egg antigen（SEA）The nano antibody of epitope, while also disclose the gene order for encoding the nano antibody and the host cell for expressing the nano antibody.The nano antibody gene order and host cell, the nano antibody announced by the present invention being capable of high efficient expression, the research and development applied to blood fluke detection reagent in Escherichia coli.
Description
Technical field
The present invention relates to biotechnology or biomedical sector, it is related to one kind and is directed to Schistosoma japonicum soluble egg and resist
It is former（Soluble Egg Antigen, SEA）Nano antibody, its coded sequence and application.
Background technology
Blood fluke is distributed widely in all over the world, especially developing country.There are five kinds of blood flukes to infect human body simultaneously
Cause the snail fever of human body.In China based on Schistosoma japonicum, the deposition of schistosoma japonice ovum can cause the stifled of blood vessel
Plug, necrosis, granuloma reaction and tissue fibrosis, therefore worm's ovum has in Schistosoma japonicum is to the pathogenic course of human body
Important effect.Research for snail fever, it is two main aspects to develop preferable diagnostic reagent and vaccine.Initially, blood
Etiological examination of the diagnostic method of fluke disease based on the inspection of conventional excrement, this method is because workload is big, loss is high, it is impossible to full
The needs of sufficient epidemic situation detection；It is one of main method of Diagnosis of Schistosomiasis that egg antigen, which is used for clinical detection, at present.Dunne D
Deng《U.S.'s tropical medicine and hygienic magazine》(Am J Trop Med Hyg 1988；38:508) reported in, by soluble worm
Ovum antigen (SEA) is isolated and purified to obtain purified fragments egg antigen with cation-exchange chromatography, and the egg antigen purified is
Cause one of important component caused by ring ovum deposited phenomenon, be currently used primarily in the diagnosis of Manson's schistosomiasis.But worm at present
Ovum antigen, come what is prepared, will so expend substantial amounts of animal mainly with experimental animal, therefore prepare cost height, and preparation method
It is more loaded down with trivial details.
Nano antibody technology, be on the basis of conventional antibodies, research and develop to obtain with Protocols in Molecular Biology, be at present
Know the antibody molecule with reference to antigen of minimum.Initially there is Belgian scientist Hamers.R to be found in camel blood, commonly
Antibody protein be made up of two heavy chains and two light chains, and the novel antibodies found from camel blood only have two heavy chains,
There is no light chain, these novel antibodies can combine closely as normal antibody in antigen, but mutually viscous unlike single-chain antibody
Company assembles blocking.Not only molecular weight is the 1/10 of common antibody to the nano antibody built based on it, and chemical property
More flexibly, stability is good, and soluble high, expression is easy and is readily available, and can be coupled other molecules, therefore applies nanometer
Antibody technique research and development blood fluke detection reagent has broad application prospects.The present invention is exactly to utilize the technology, using screening
The special nano antibody of the schistosoma japonice ovum antigen that arrives, for detecting infection by Schistosoma.
The content of the invention
The purpose of the present invention is in view of the shortcomings of the prior art, there is provided one kind is directed to Schistosoma japonicum soluble egg antigen
（SEA）Nano antibody, while provide the nano antibody coded sequence and the nano antibody prepare detection application.
The purpose of the present invention is achieved through the following technical solutions：A kind of VHH chains of nano antibody for SEA, bag
Including framework region FR and complementary determining region CDR, the framework region FR includes FR1~FR4 amino acid sequence：Wherein, FR1 amino
Acid sequence such as SEQ ID NO：Shown in 1, FR2 amino acid sequence such as SEQ ID NO：Shown in 3, FR3 amino acid sequence
Such as SEQ ID NO：Shown in 5, FR4 amino acid sequence such as SEQ ID NO：Shown in 7；The complementary determining region CDR includes
CDR1~CDR3 amino acid sequence：CDR1 amino acid sequence SEQ ID NO：Shown in 2, CDR2 amino acid sequence SEQ
ID NO：Shown in 4, CDR3 amino acid sequence SEQ ID NO：Shown in 6.
Further, the VHH chains of described SEA nano antibody, it has SEQ ID NO：Amino acid sequence shown in 9
Row.
A kind of SEA nano antibodies, it is directed to the nano antibody of SEA epitopes, including two have SEQ ID NO：Ammonia shown in 9
The VHH chains of base acid sequence.
A kind of DNA molecular, it encodes the protein being selected from the group：The VHH chains of the above-mentioned nano antibody for SEA, or
Above-mentioned SEA nano antibodies.
A kind of expression vector, it contains SEQ ID NO：Nucleotide sequence shown in 8.
A kind of host cell, it can express SEA nano antibodies.
A kind of application of SEA nano antibodies in terms of SEA detection reagents are prepared.
Beneficial effect：Compared with prior art, advantages of the present invention is as follows：The method comprises the steps of firstly, preparing and the Japanese blood of purifying inhale
Worm egg antigen, is immunized camel, prepares the special nano antibody storehouses of SEA, SEA after purification then is coupled at into ELISA Plate
On, the special nano antibodies of the high SEA expressed are screened using display technique of bacteriophage, and e. coli tg1 is transferred to, establish
Can in e. coli tg1 high efficient expression nano antibody strain.
Brief description of the drawings
Fig. 1 is the DNA electrophoretograms of nano antibody, and M molecular weight marker, 1 PCR primer, band about 600bp, 1-13 are
SEA nano antibodies；N is negative control；
Fig. 2 is the special nano antibodies of blood fluke SEA, is purified again through AKTAexpress after purification through affinity chromatography
SDS-PAGE afterwards（A）With Western blot trace figures（B）, wherein M is molecular weight marker, unit kD, and 1-6 is represented
SEA nano antibodies；
Fig. 3 is the EUSA using SEA nano antibodies identification blood fluke SEA（ELISA）As a result.In figure
" ▲ " curve is SEA nano antibodies, and " ■ " curve is negative control antibody VSG.
Embodiment
Following examples are used for illustrating the present invention, rather than limit the invention, the present invention spirit and
In scope of the claims, to any modifications and changes of the invention made, protection scope of the present invention is both fallen within.
Present invention application is coupled on ELISA Plate for SEA nano antibodies, the correct space conformation of display protein matter, with this
The antigen selection immune nano antibody library of form（Camel heavy chain antibody phage display library）, and obtain can be in Escherichia coli
The nano antibody strain of high efficient expression.
With reference to specific embodiment, the present invention is expanded on further.
Embodiment 1
The special nano antibody storehouses of the present embodiment structure Schistosoma japonicum SEA, step are as follows：
（1）Schistosoma japonicum SEA is purified first, then mixes 1mg SEA antigens in equal volume with Freund's adjuvant, is immunized one
Alpaca（Alpa-Vet, www.alpa-vet.be）, once in a week, it is immunized 7 times altogether, stimulates B cell to express antigentic specificity
Nano antibody；
（2）After 7 immune end, extract 100ml alpacas PBLC and simultaneously extract total serum IgE；
（3）Synthesize cDNA and utilize sleeve type PCR amplification VHH；
（4）Utilize restriction enzyme pstI and NotI digestion 20ug Vector for Phage Display and 10ulVHH and connection two
Individual fragment；
（5）Connection product is converted to electricity and turned in competent cell TG1, SEA nano antibodies library is built and determines storage capacity,
Storage capacity size is 8.9 × 109。
Embodiment 2
Nano antibody special the present embodiment screening SEA, step are as follows：
（1）It is dissolved in 100mM NaHCO3, pH8.2 20ug SEA be coated on NUNC ELISA Plates, 4 DEG C were placed
Night；
（2）Add within second day 100ul 3%milk, room temperature closing 2h；
（3）After 2h, 100ul 2 × 10 is added11Tfu contains the helper phage in SEA nano antibodies library, room temperature effect
1h；
（4）First round elutriation washes the 10 times/second 20-25 times/third round of wheel 20 times with 0.05%PBS+Tween-20, removes
The bacteriophage of non-specific binding；
（5）Under being dissociated with 100mM TEA (triethylamine) with the bacteriophage of SEA specific bonds, and infect and be in
The e. coli tg1 of exponential phase, 37 DEG C of culture 1h, produce the screening that simultaneously purified phage is used for next round, identical screening
Process repeats 3-4 wheels, is progressively enriched with.
Embodiment 3
The present embodiment enzyme-linked immunoassay method of bacteriophage（ELISA）The single positive colony of screening specificity, step are as follows：
（1）From the Tissue Culture Dish containing bacteriophage after above-mentioned 3-4 wheel screenings, select 96 single bacterium colonies and be inoculated in
In the TB culture mediums of ampicillin containing 100ug/ml, after growing into logarithmic phase, add final concentration 1mM IPTG, 28 DEG C of trainings
Support overnight.
（2）Obtained using osmosis and slightly carry antibody, and antibody is transferred in the elisa plate through antigen coat, at room temperature
Place 1 hour；
（3）Uncombined antibody is washed away with PBST, adds mouse anti-His antibody (the anti-HIS antibody of mouse, R＆D
System), place 1 hour at room temperature；
（4）Uncombined antibody is washed away with PBST, adds anti-mouse alkaline phosphatase
Conjugate (sheep anti mouse AP labelled antibodies, sigma).
（5）Uncombined antibody is washed away with PBST, adds alkaline phosphatase nitrite ion, 405nm wavelength is read on ELIASA
Take absorption value.
（6）When sample well OD values are more than more than 2.1 times of control wells OD values, it is judged as positive colony hole.
（7）The bacterium in positive colony hole is turned to shake the TB culture mediums in the ampicillin containing 100ug/ml, extracts plasmid
It is sequenced.
According to sequencing result, using Vector NTI®11.5 (Invitrogen, USA) and IMGT®Software analysis is each
Individual clone strain, CDR1, CDR2 and CDR3 sequence identical strain are considered as same clone strain, and sequence it is different be considered as different clones
Strain.The nucleotide sequence of the VHH chains for the specific SEA nano antibodies established is screened as shown in SEQ ID NO .8, amino acid
Sequence is as shown in SEQ ID NO .9.The amino acid sequence of VHH chains, it is made up of framework region FR and complementary determining region CDR, the frame
Frame area FR includes SEQ ID NO：FR1 shown in 1, SEQ ID NO：FR2 shown in 3, SEQ ID NO：FR3 shown in 5,
SEQ ID NO：FR4 shown in 7；The complementary determining region CDR includes SEQ ID NO：CDR1 shown in 2, SEQ ID NO：4
Shown CDR2, SEQ ID NO：CDR3 shown in 6.Wherein, FR1~FR4 and CDR1~CDR3 sequence is as follows：
FR1：QVQLQESGGGLVQAGASLRLSCATSA；
CDR1：RTFNSYSMKWFR；
FR2：QAPGKEREFVARISRSG；
CDR2：GTTYYADSVK；
FR3：GRFTISRDTAKSVVYLQMNSLKPEDTAIYY；
CDR3：CAAAIFDVTDY；
FR4：ERADYWGQGTQVTVSS；
Nucleotide sequence and amino acid sequence based on VHH chains, can also obtain following product：
A kind of SEA nano antibodies, it is directed to the nano antibody of SEA epitopes, including two have SEQ ID NO：Ammonia shown in 9
The VHH chains of base acid sequence.
A kind of DNA molecular, it encodes the protein being selected from the group：The VHH chains of the above-mentioned nano antibody for SEA, or
Above-mentioned SEA nano antibodies.
A kind of expression vector, it contains SEQ ID NO：Nucleotide sequence shown in 8.
A kind of host cell, it can express SEA nano antibodies.
Embodiment 4
The specific SEA nano antibodies obtained using screening carry out enzyme linked immunosorbent detection, and step is as follows：
Obtained by the detection specific SEA nano antibodies of screening whether can identify SEA antigens, by SEA antigens (2 μ g/
ML 96 hole elisa Plates) are added, 100 μ L/ holes, 4 °C overnight；With PBST board-washings 3 times, after with 3%milk close ELISA Plate, 37 °C
Lh is acted on, with PBST board-washings 3 times, adds the SEA nano antibodies of different diluted concentrations, 37 °C of effect 2h；After PBST board-washings 3 times,
Add horseradish peroxidase（HRP）The anti-His monoclonal antibodies of mouse (AbD Serotec, UK) the effect 1h of mark；PBST is washed
After plate 3 times, add tmb substrate liquid after detect, and with build trypanosome specificity VSG nano antibodies be used as negative control.
As Fig. 3 is shown, SEA nano antibodies can with specific recognition SEA, and negative control trypanosome VSG nano antibodies then with
SEA is reactionless, illustrates that SEA nano antibodies proposed by the present invention can further develop detection SEA reagent.
Described above is only the preferred embodiment of the present invention, under the premise without departing from the principles of the invention, can also be to this
Improvements and modifications are made in invention, and these improvements and modifications also should be regarded as protection scope of the present invention.
SEQUENCE LISTING
<110>Zhejiang Academy of Medical Sciences
<120>It is a kind of for SEA nano antibody and its coded sequence and application
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> PRT
<213>It is artificial synthesized
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Ala
20 25
<210> 2
<211> 12
<212> PRT
<213>It is artificial synthesized
<400> 2
Arg Thr Phe Asn Ser Tyr Ser Met Lys Trp Phe Arg
1 5 10
<210> 3
<211> 17
<212> PRT
<213>It is artificial synthesized
<400> 3
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Arg Ile Ser Arg Ser
1 5 10 15
Gly
<210> 4
<211> 10
<212> PRT
<213>It is artificial synthesized
<400> 4
Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10
<210> 5
<211> 30
<212> PRT
<213>It is artificial synthesized
<400> 5
Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Ser Val Val Tyr Leu
1 5 10 15
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr
20 25 30
<210> 6
<211> 11
<212> PRT
<213>It is artificial synthesized
<400> 6
Cys Ala Ala Ala Ile Phe Asp Val Thr Asp Tyr
1 5 10
<210> 7
<211> 16
<212> PRT
<213>It is artificial synthesized
<400> 7
Glu Arg Ala Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10 15
<210> 8
<211> 366
<212> DNA
<213>It is artificial synthesized
<400> 8
caagttcagc tccaagagtc gggcggaggc ctggttcaag cgggagccag cctcaggctc 60
agctgtgcca cgagcgcccg gaccttcaat agctatagca tgaagtggtt caggcaggct 120
ccaggcaagg agagagagtt tgtcgcaagg ataagcagga gcggcgggac aacgtactac 180
gccgatagcg ttaagggccg cttcacgatc agccgggaca ccgcgaagac ggttgtctac 240
ctgcagatga atagcttaaa acccgaggat acggctatat actactgcgc cgcggcaata 300
tttgatgtca cagattacga gcgagcggac tactggggcc aaggtacgca agtcactgtc 360
agcagc 366
<210> 9
<211> 122
<212> PRT
<213>It is artificial synthesized
<400> 9
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Ala Arg Thr Phe Asn Ser Tyr
20 25 30
Ser Met Lys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Arg Ile Ser Arg Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Ser Val Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Ala Ile Phe Asp Val Thr Asp Tyr Glu Arg Ala Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
Claims (7)
1. a kind of VHH chains of nano antibody for SEA, including framework region FR and complementary determining region CDR, it is characterised in that institute
Stating framework region FR includes FR1~FR4 amino acid sequence：Wherein, FR1 amino acid sequence such as SEQ ID NO：Shown in 1, FR2
Amino acid sequence such as SEQ ID NO：Shown in 3, FR3 amino acid sequence such as SEQ ID NO：Shown in 5, FR4 amino
Acid sequence such as SEQ ID NO：Shown in 7；The complementary determining region CDR includes CDR1~CDR3 amino acid sequence：CDR1 ammonia
Base acid sequence SEQ ID NO：Shown in 2, CDR2 amino acid sequence SEQ ID NO：Shown in 4, CDR3 amino acid sequence SEQ
ID NO：Shown in 6.
2. VHH chains according to claim 1, it is characterised in that it has SEQ ID NO：Amino acid sequence shown in 9.
3. a kind of SEA nano antibodies, it is characterised in that it is directed to the nano antibody of SEA epitopes, including two have SEQ ID
NO：The VHH chains of amino acid sequence shown in 9.
4. a kind of DNA molecular, it is characterised in that it encodes the protein being selected from the group：SEA nanometers described in claim 1 or 2
The VHH chains of antibody, or the SEA nano antibodies described in claim 3.
5. a kind of expression vector, it is characterised in that it contains SEQ ID NO：Nucleotide sequence shown in 8.
6. a kind of host cell, it is characterised in that it can express the SEA nano antibodies described in claim 3.
A kind of 7. application of the SEA nano antibodies in terms of SEA detection reagents are prepared described in claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410795870.2A CN104650224B (en) | 2014-12-22 | It is a kind of for SEA nano antibody and its coded sequence and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410795870.2A CN104650224B (en) | 2014-12-22 | It is a kind of for SEA nano antibody and its coded sequence and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104650224A CN104650224A (en) | 2015-05-27 |
| CN104650224B true CN104650224B (en) | 2018-02-09 |
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