CN102690336A - Bat SARS-like coronavirus spike protein immunity determining area and preparation method and application thereof - Google Patents

Abstract

The invention discloses a bat SARS-like coronavirus spike protein immunity determining area and a preparation method and application thereof. The method comprises the following steps of: A, amplifying by taking the bat SARS-like coronavirus S gene as a template design primer; B, performing enzyme digestion of the fragment obtained by the amplification by use of BamHI and XhoI, connecting to an expression vector pET32a, and sequencing to assure correctness; and C, purifying the recombinant plasmid, transforming the BL21 competent cell, culturing the monoclonal antibody, and performing 30-degree induction in a culture medium with the final concentration of 0.3mMIPTG; collecting the thalli, performing ultrasonic breaking, and then purifying with a HisTag purification kit; and after the purification, detecting the concentration of protein in SDS-PAGE to obtain the target protein. The method is simple and easy to implement, convenient to operate and easy in production; the peptide has the best immunogenicity; and a method of applying the protein to the identification of mice S monoclonal antibody epitope has high specificity, and is simple to operate and easy to repeat.

Description

Bat SARS appearance coronavirus spike protein immunity determining area and preparation method and purposes

Technical field

The invention belongs to biological technical field; More specifically relate to the immune determining area of a kind of bat SARS appearance coronavirus spike protein (Rp3-S); The preparation method who also relates to the immune determining area of a kind of bat SARS appearance coronavirus spike protein (Rp3-S) simultaneously also relates to the purposes of the immune determining area of a kind of bat SARS appearance coronavirus spike protein (Rp3-S).

Background technology

The cause of disease of the sars coronavirus SARS that to be 2002-2003 break out in China.And there are very high homology in bat SARS appearance coronavirus and sars coronavirus.After being found first by this seminar in 2005,, but few in the world wide about the immunogenic report of bat SARS appearance coronavirus all relevant for the report of similar virus.Hong Kong University thinks that through information science this virus has escape and propagates the danger in the mankind.Research shows that the main difference of bat SARS appearance coronavirus and sars coronavirus is at S albumen, and promptly main being responsible for combines, induces body to produce the albumen of neutralizing antibody with cell receptor.Bat SARS appearance coronavirus just can be caused a disease to mouse as long as obtain a bit of on people's sars coronavirus S albumen.This shows that this virus has potential pathogenic.Thereby, about the research of this virus immunity originality for designs specificity vaccine or the diagnostic method particularly important that just seems.

Applicant's work is in earlier stage found; The bat serum of bat SARS appearance coronavirus infection and people's sars coronavirus S albumen have cross reaction; But can not in people's sars coronavirus, explain between bat SARS appearance coronavirus and the people's sars coronavirus S albumen to exist very big difference.This can be owing to the part of two virus S proteins and receptors bind, and the zone of promptly inducing body to produce neutralizing antibody exists relatively low homology (Fig. 1).

This study group in the work in early stage, utilize pseudovirus invasion experiment confirm the proteic immune determining area of bat SARS appearance coronavirus S be positioned at the corresponding zone of its receptor binding domain.Confirming for the research and development of design bat SARS appearance coronavirus specific diagnostic, protectiveness vaccine and antiviral of the particular location of this immunity determining area is very important.

Summary of the invention

The objective of the invention is to be to provide a kind of bat SARS appearance coronavirus (Rp3) spike protein (Rp3-S) immune determining area albumen, its aminoacid sequence is shown in the SEQ ID NO.1.This determining area information can be used for the specific diagnosis of bat SARS appearance coronavirus for identifying first.

Another object of the present invention is to be to provide the immune determining area of a kind of bat SARS appearance coronavirus spike protein (Rp3-S) proteic preparation method.This method is simple, and is easy to operate, is easy to produce.

A further object of the present invention is to be to provide the application of the S280-455 polypeptide fragment in a kind of bat SARS appearance coronavirus spike protein (Rp3-S) in identifying mouse anti S monoclonal antibody epi-position.This method specificity is good, simple to operate, and experiment is easy to repetition.

To achieve these goals, the present invention adopts following technical measures:

Immune determining area albumen of a kind of bat SARS appearance coronavirus spike protein (S) and preparation method thereof, it may further comprise the steps:

According to the work in early stage of this study group, the applicant has compared the difference of bat SARS appearance coronavirus and SARS-CoV S protein immunization originality, recognizes that the difference of their immunodominant regions possibly be its receptor binding domain.Yet the applicant does not still know the concrete amino acid position of its immunodominant region.For this reason; The applicant is with S gene (GenBank sequence number: DQ071615; Position at genome nucleotide sequence:: 21486-25211bp) sequence is cut into 6 sections (Fig. 2); Carry out the protokaryon protein expression respectively, total length S monoclonal antibody clonal antibody, the polyclonal antibody of then having used the mouse source are identified the particular location of S immunity determining area.

The segmental pcr amplification that six of A.Rp3-S block:

Rp3-S proteic S1 district and five zones blocking thereof are respectively: 1-666aa, expressed proteins called after S1-666, nucleotides sequence classify as shown in the SEQ ID NO.2; 1-81aa, expressed proteins called after S1-82 nucleotides sequence is classified as shown in the SEQID NO.3; 82-280aa, marking protein called after S82-280, nucleotides sequence classify as shown in the SEQ ID NO.4; 280-455aa, expressed protein called after S280-455, nucleotides sequence classify as shown in the SEQ ID NO.5; 429-574aa, expressed protein called after S429-574, nucleotides sequence classify as shown in the SEQ ID NO.6; 561-666aa, protein called after S561-666, nucleotides sequence classify as shown in the SEQ ID NO.7.

According to six segmental nucleotide sequences, 6 pairs of primers of design are following:

1-666: forward: 5 '-GGCCGAATTCATGAAAATTTTAAT-3 ',

Oppositely: 5 '-CAGTGTCGACTTAAGACATAGTGTAAGCCAC-3 ',

1-81aa: forward: 5 '-GGCCGGATCCATGAAAATTTTAAT-3 ',

Oppositely: 5 '-ACTTGTCGACGTAGGTAAACCTAT-3 ';

82-280aa: forward: 5 '-GGCCGGATCCTTTGACAATCCTAT-3 ',

Oppositely: 5 '-CTCAGTCGACAATAGCATTGGTAA-3 ';

280-455aa: forward: 5 '-TGCCGGATCCATTGATTGTGCTCA-3 ' is SEQ ID NO.8,

Oppositely: 5 '-CAGTGTCGACTTAAGAAAGGTCTCTCTCAAA-3 ' is SEQ IDNO.9;

429-574aa: forward: 5 '-GGACGGATCCACTGCAAAGCAGGATCAA-3 ',

Oppositely: 5 '-GAACGTCGACTGTTCCTGGTGTAATTAC-3 ';

561-666aa: forward: 5 '-GGCTGGATCCCCATGCTCTTTTGG-3 ';

Oppositely: 5 '-CAGTGTCGACTTAAGACATAGTGTAAGCCAC-3 '.

The reagent of PCR reaction is following: with the 10xTaq polymerase buffer of 5 μ l, the dNTP mixture of 4 μ l, the TaqDNA polysaccharase of 0.25 μ l, plasmid pCDNA3.1-Rp-S (Ren, W., Qu, X., the Li that 1 μ l contains the Rp3-S gene; W., Han, Z., Yu; M., Zhang, S., Wang; L.F., Deng, H., Shi; Z.Difference in receptor usage between SARScoronavirus and SARS-like coronavirus of bat origin.J Virol, 2008,82 (4): 1899 – 1907), primer mixes each sterilized water with 1 μ l and 37.75 μ l.The PCR reaction conditions: 30 seconds, 72 ℃ of 30 seconds, 56 ℃ renaturation of 300 seconds, 94 ℃ sex change of 94 ℃ of preparatory sex change were extended 60 seconds, and 72 ℃ of last extensions 600 seconds circulates after 30 times.

B. vector construction:

Be connected in expression vector pMAL-C2X (available from New England Biolabs) after cutting through BamHI and XhoI enzyme through the Rp3-S1 that obtains after the above-mentioned amplification, five are blocked and are connected to expression vector pET32a after fragment is cut through BamHI and XhoI enzyme and go up (available from Novagen).All structure plasmids all pass through order-checking and confirm errorless.

C. proteic expression and purifying:

Behind the recombinant plasmid purifying, transform BL21 (available from Promega) competent cell, be incubated at again and contain on penbritin (Amp) resistant panel.Next day, the picking mono-clonal was cultivated, then final concentration be in the substratum of 0.3mMIPTG 30 ℃ induced 6 hours.Carry out purifying after collecting the thalline ultrasonic disruption.S1 protein requirement with the pMALc2x vector expression carries out purifying with the MBP resin column, and with having His Tag label on the pET32a expression vector, expressed proteins is all carried out purifying with His Tag purification kit.All purification steps all carry out according to the His-band test kit explanation of Promega company.In SDS-PAGE, detect proteic purity (Fig. 3) behind the purifying, promptly obtain target protein.All purity of protein have all reached more than 90%.

D. the preparation of the mice serum of bat SARS appearance coronavirus S dna immunization:

PCDNA3.1-Rp3-S is injected female BALB/c mouse in 6-8 age in week with the mode that shocks by electricity in the body; Respectively 0,3 and 5 when week immune mouse; The 8th week, extract mice serum, these serum are the antiserum(antisera) that contains bat SARS appearance coronavirus S protein polyclone antibody.The serum portion of adopting healthy mice simultaneously is as negative control, totally five parts of serum samples, and the positive serum sample is called after Rp-S1 ~ Rp-S4 successively.

E. the proteic establishment of the immune determining area of a bat SARS appearance coronavirus spike protein (Rp3-S) the steps include:

At first with 5 purifying block S1 albumen as envelope antigen, mouse anti S albumen serum is anti-as one, the sheep anti mouse of HRP mark two is anti-to carry out the ELISA reaction.The result shows 5 mice serums and 5 albumen tests different (Fig. 4).

The result shows; Most of immune serums can react with S280-455 and S561-666: four parts of immune serums of Rp-S1 ~ Rp-S4 all can react with S280-455; Rp-S1; Rp-S3 and Rp-S4 and S561-666 reaction have only a serum of Rp-S2 and S429-574 reaction, do not have serum to react with S1-82 and S82-455.In view of the S561-666 section exists immunodominant region very conservative in the coronavirus; Thereby can not be as the epi-position of the special detection of bat SARS appearance coronavirus; And S280-455 exists one of bat SARS appearance coronavirus and people's sars coronavirus S albumen key distinction; Thereby the applicant think that S280-455 exists can be by mouse antibodies identification and the main immunity decision epi-position that can be employed, its aminoacid sequence is shown in the SEQ ID NO.1.

This peptide section has best immunogenicity; It is the proteic main immunity of S district; And should the zone and the proteic main immune determining area of people's sars coronavirus S and not exclusively overlapping; This finds to be directed against for structure the special detection method of bat SARS appearance coronavirus, and designs to this viral vaccine and all want important meaning, in the special evaluation work of following this viroid, has good prospect.

The application of a kind of bat SARS appearance coronavirus spike protein immunity determining area albumen in identifying mouse anti S monoclonal antibody epi-position the steps include:

A. mouse anti bat SARS appearance coronavirus S MONOCLONAL ANTIBODIES SPECIFIC FOR: the applicant will contain the bat SARS appearance coronavirus Rp3 proteic pseudovirus of strain S (HIV/Rp3-S) as antigen in BALB/c mouse according to standard method (Evan; G.Iet al; 1985, Isolation of monoclonal antibodies specific for human c-myc proto-oncoge ne product.Mol Cell Biol 5:3610-6.) preparation.To select male clone's enlarged culturing and abdominal injection and go into, and collect ascites with the source mouse.It is tired and identifies through ELISA subsequently.For fear of the cross reaction of these monoclonal antibodies and carrier pET32a tag (available from Novagen), before using, all monoclonal antibodies all neutralize with the bacterial lysate that contains pET32a tag.

B. identify the protein hybridization reaction of mouse anti S monoclonal antibody with S280-455.As antigen, mouse anti S monoclonal antibody is carried out the protein hybridization reaction as detecting antibody with S280-455 albumen.Screen four S protein monoclonal antibodies, name SmAb1-SmAb4, identification S280-455 that all can be special rather than pET32a Tag or other four the S albumen that block expression.It is main immune determining area (Fig. 5) that these data have been confirmed S280-455.

The present invention compared with prior art has the following advantages and effect:

At first; The main immune determining area S280-455 of bat SARS appearance coronavirus S albumen that identifies, this peptide section has best immunogenicity, is the proteic main immunity of S district; And should the zone and the proteic main immune determining area of people's sars coronavirus S and not exclusively overlapping; This finds to be directed against for structure the special detection method of bat SARS appearance coronavirus, and designs to this viral vaccine and all want important meaning, in the special evaluation work of following this viroid, has good prospect;

Secondly, the proteic preparation method of the immune determining area of bat SARS appearance coronavirus spike protein (S) who provides, this method is simple, and is easy to operate, is easy to produce;

Once more, the application method specificity of the S280-455 polypeptide fragment that provides in identifying mouse anti S monoclonal antibody epi-position is good, simple to operate, is easy to repetition.

Description of drawings

Fig. 1 is that a kind of bat SARS appearance coronavirus Rp3-S is corresponding to the proteic receptor binding domain synoptic diagram of sars coronavirus Tor2-S.

Numeral is an amino acid sites among the figure.The underscore part is both difference the best parts, also is Tor2-S albumen and receptors bind core.

Fig. 2 is bat SARS appearance coronavirus S1 part and five S fragment synoptic diagram that block of a kind of total length.

The SDS-PAGE synoptic diagram of Fig. 3 after for a kind of Rp3-S proteic S1 district and five SARS appearance coronavirus S protein purifications that block thereof.Albumen all has the label of 20kD.

Fig. 4 is can be by the immunodominant region synoptic diagram of mouse S antibody recognition on the bat SARS appearance coronavirus S1.

The S albumen that blocks expression is as envelope antigen, mice serum (called after Rp-S1 is to the Rp-S4) reaction of they and four parts of Rp3-S dna immunizations.A normal mouse serum is as negative control.Data are expressed as mean+/-standard error.

Fig. 5 is a kind of epi-position synoptic diagram of tiring and being discerned of the Rp3 of evaluation S1 mouse monoclonal antibody.

In the protein hybridization reaction, have only S280-455 to be discerned by monoclonal antibody.Monoclonal antibody 1:250 dilutes use.Numeral among the figure: 1, pET32a tag albumen; 2, S280-455 albumen.

Embodiment

Embodiment 1: design amplification bat SARS appearance coronavirus S gene blocks fragment, the steps include:

Sequence (the GenBank sequence number: DQ071615 of the bat SARS appearance coronavirus S gene that obtains based on this laboratory previous work; Position at genome nucleotide sequence: 21486-25211), design primer respectively and carry out pcr amplification and obtain Rp3-S1 and five goal gene that block S thereof.Primer is distinguished as follows:

1-666: forward: 5 '-GGCCGAATTCATGAAAATTTTAAT-3 ',

Oppositely: 5 '-CAGTGTCGACTTAAGACATAGTGTAAGCCAC-3 ';

1-81aa: forward: 5 '-GGCCGGATCCATGAAAATTTTAAT-3 ',

Oppositely: 5 '-ACTTGTCGACGTAGGTAAACCTAT-3 ';

82-280aa: forward: 5 '-GGCCGGATCCTTTGACAATCCTAT-3 ';

Oppositely: 5 '-CTCAGTCGACAATAGCATTGGTAA-3 ';

280-455aa: forward: 5 '-TGCCGGATCCATTGATTGTGCTCA-3 ',

Oppositely: 5 '-CAGTGTCGACTTAAGAAAGGTCTCTCTCAAA-3 ';

429-574aa: forward: 5 '-GGACGGATCCACTGCAAAGCAGGATCAA-3 ',

Oppositely: 5 '-GAACGTCGACTGTTCCTGGTGTAATTAC-3 ';

561-666aa: forward: 5 '-GGCTGGATCCCCATGCTCTTTTGG-3 ',

Oppositely: 5 '-CAGTGTCGACTTAAGACATAGTGTAAGCCAC-3 '.

Each carries out pcr amplification with above-mentioned a pair of primer each gene.The reagent of PCR reaction is following: 10xTaq polymerase buffer, the dNTP mixture of 4 μ l, the TaqDNA polysaccharase of 0.25 μ l, 1 μ of 5 μ l are contained the pcDNA3.1-Rp3-S plasmid, and primer mixes each sterilized water with 1 μ l and 37.75 μ l.The PCR reaction conditions: 30 seconds, 72 ℃ of 30 seconds, 56 ℃ renaturation of 300 seconds, 94 ℃ sex change of 94 ℃ of preparatory sex change were extended 60 seconds, and 72 ℃ of last extensions 600 seconds circulates after 30 times.

Embodiment 2:

Structure and the expression and the purifying of bat SARS appearance coronavirus S truncated protein recombinant expression vector the steps include:

With restriction enzyme BamHI and XhoI (Takara Company products) the PCR product that reclaims and expression vector pET32a plasmid being carried out enzyme cuts.Each l μ l of endonuclease reaction: BamHI and XhoI, 10 times of H damping fluid 2 μ l,, PCR product or pET32a plasmid plasmid 50-100ng, adding sterilized water to TV is 20 μ l.37 ℃ 1 hour, enzyme is cut product and after DNA reclaims, with T4DNA ligase enzyme (Takara Company products) the PCR product is connected with expression vector pET32a.Ligation: T4DNA ligase enzyme (1U/ μ l) l μ l, the mol ratio of PCR product and expression vector pET32a is 3:1, and the DNA total amount is 0.1 μ g, 5 times of ligase enzyme reaction buffer 4 μ l, adding sterilized water to TV is 20 μ l, places 24 hours for 16 ℃.

2 μ l ligation liquid are added to the DH5 α competent cell of 60 μ l, mixing, electric shock transforms, 37 ℃ of shaking tables (220rpm) incubation 1 hour; Shake up bacterium liquid, get 100 μ l and coat LB/AP ⁺On the agar plate, treat to be inverted in after bacterium liquid blots 37 ℃ and cultivated observations 12～16 hours.Next day, picking list bacterium colony was 10, contained 37 ℃ of overnight cultures in the LB liquid nutrient medium of ammonia benzyl in 4ml.Treat that bacterium liquid cultivate to finish, get bacterium liquid and extract plasmid and carry out enzyme with BamHI and XhoI and cut evaluation.There is segmental positive colony of insertion to send order-checking company to carry out sequencing analysis.

Select a kind positive monoclonal, inoculate fresh Amp LB substratum.250rpm, 37 ℃, the concussion overnight cultures.The 3rd day, with incubated overnight bacterium liquid,, inoculate fresh LB substratum in the 1:100 ratio, add the Amp resistance equally.In 37 ℃, 250rpm concussion was cultivated 3-4 hour, reached OD260=0.6～1.0 to bacterial concentration, and this moment, bacterium reached logarithmic phase.The adding final concentration is that the IPTG of 0.3mM carries out abduction delivering.250rpm, continues concussion and cultivated centrifugal results thalline 6 hours by 30 ℃.Behind twice of PBS solution washing, be resuspended in (ratio of concentrating is that the original bacterium liquid of 200ml is in 20mlPBS) among an amount of 0.01mol/L PBS.Behind-80 ℃ of multigelations three times, carry out ultrasonic disruption and protein purification.Because have His Tag label on the pET32a expression vector, so expressed proteins can use His Tag purification kit (Hisband is available from Novagen) to carry out purifying.Purge process is following: at first inducing cell is carried out ultrasonic disruption, afterwards, and 16000rpm, 4 ℃, centrifugal 15min collects supernatant, filters with 0.45nm aperture filter.Next use His Bind purification kit purifying, key step comprises: at first, add 3 times distilled water successively, and 5 times Charge Buffer, 3 times Bi nding Buffer carries out balance to pillar; Supernatant after filtering is passed through pillar; The Binding Buff er that adds 10 times successively, 6 times Wash Buffer washing; Carry out the albumen wash-out with 6 times Elute Buffer at last.After all purifying finishes, His Bind purifying pillar is regenerated.The albumen that finally is purified into all through SDS-PAGE, determination of protein concentration guarantee purity reach more than 95% and concentration greater than 100ng/ μ l (Fig. 3).

The protein called after S1-82 that 1-81aa obtains after expressing; The protein that 82-280aa obtains after expressing is S82-280; The protein called after S280-455 that 280-455aa obtains after expressing; The protein called after S429-574 that 429-574aa obtains after expressing; The protein called after S561-666 that 561-666aa obtains after expressing.

Embodiment 3:

Bat SARS appearance coronavirus S blocks the ELISA reaction of the mice serum of fragment and S dna immunization.

The serum of mouse anti total length S comes from the following plasmid of dna immunization: Rp3-S (the Ren et al that is implemented in the codon optimized mistake of pCDNA3.1 (+); 2008; Journal of Virology, Difference in Receptor Usage between Severe Acute Respiratory Syndrome (SARS) Coronavirus and SARS-Like Coronavirus of Bat Origin)) and pCDNA3.1 (+) empty carrier plasmid (Introgen company) be used as negative control.30 μ g plasmids are injected female BALB/c mouse in 6-8 age in week with the PBS dilution back of 30 μ l with the mode that shocks by electricity in the body; Experiment is divided into and is 2 groups and (sees zhou et al for details; 2009; BBRC, Immunogenicity difference between the SARS coronavir us and the batSARS-like coronavirus spike (S) proteins), every group has 5 mouse.Immune mouse when 0,3 and 5 weeks is got mice serum the 8th week respectively.5 by Rp3-S mice immunized serum called after Rp-S1 ~ Rp-S4, and Pre-bleed is a mice serum control group before the immunity.

After making antibody, the applicant carries out enzyme linked immunoassay and measures the reactivity of expressing fragment and antibody.Method is following: at first encapsulate 96 hole enzyme plates with purified proteins, every hole adds 100 μ l substrate coated elisa plates, and 4 ℃ are spent the night.Next day, take out with PBS-T (containing volume ratio and be the PBS of 0.1% Tween-20) and wash each 5 minutes three times.Then, add 3% (mass volume ratio) bovine serum albumin, the every hole of 200 μ l, 37 ℃, left standstill 1 hour, seal.After 1 hour, take out PBS-T washing three times, each 5 minutes.Carry out sero-fast detection to be measured after the washing.Every hole adds test serum, and final volume is 100 μ l.Simultaneously, do the negative control group of normal serum and PBS solution.37 ℃, hatch 1 hour after, take out response sample, PBS-T washing five times, each 3 minutes.With 1:4000 dilution proportion HRP mark sheep anti mouse two anti-(Wuhan three hawk companies), every hole adds 100 μ l.37 ℃, hatched once more 1 hour.Take out, after PBS-T washs five times once more, add substrate A (volume ratio 30% hydrogen peroxide), B (3,3,5,5, according to TMB, TMB damping fluid) each 50 μ l of liquid successively, 37 ℃, lucifuge colour developing 5 minutes.At last, every hole adds 50 μ l 2M/L H ₂SO ₄Termination reaction.ELIASA reads the value of OD450.The OD value is confirmed as positive reaction greater than 2.1 times of negative control hole.The result is as shown in Figure 4: most of by mice immunized serum can with S280-455 and S561-666 reaction: four parts of mice serums of Rp-S1 ~ Rp-S4 all can react with S280-455; Rp-S1; Rp-S3 and Rp-S4 and S561-666 reaction; Have only a serum of Rp-S2 and S429-574 reaction, do not have serum to react with S1-82 and S82-280.In view of the S561-666 section exists immunodominant region very conservative in the coronavirus; Thereby can not be as the epi-position of the special detection of bat SARS appearance coronavirus; And S280-455 exists one of bat SARS appearance coronavirus and people's sars coronavirus S albumen key distinction; Thereby the applicant think that S280-455 exists can be by mouse antibodies identification and the main immunity decision epi-position that can be employed, its aminoacid sequence is shown in the SEQ ID NO.1.

Embodiment 4:

The application of S280-455 in identifying S mouse monoclonal antibody identification epi-position, its application process is:

Pseudovirus (HIV/Rp3-S) preparation process to Rp3-S-is following: utilize HIVNL4-3 (Δ ENV) luc plasmid (disappearance env gene ENV; Have a luciferase reporter gene); The people SARS-CoVBJ01 of external plasmid expression; SL-CoVRp3 S albumen or RBD district are replaced by the mosaic SSL of SSARS; Substitute the proteic position of ENV, constitute HIV/BJ01-S, HIV/Rp3-S or HIV/CS310-518 pseudovirus, the infection success or not of pseudovirus can be represented with the activity of luciferase.Concrete packaging step is following:

Previous day is pressed 4x10 with the 293T cell in transfection ⁶/ ware density is inoculated in the 10cm diameter Tissue Culture Dish, renews bright substratum (perhaps changing half substratum), transfection time-division 2 1.5ml centrifuge tubes: centrifuge tube A (CaCl for before the transfection 293T cell ₂+ DNA mixture) 500 μ l (using the ultrapure water polishing) comprising: HIV-luc plasmid pNL-Luc-E-R-(Rong Liu, Benjamin Chen and Nathaniel R.Landau.Use of Luciferase Reporter Viruses for Studying HIV Entry.HIV Protocols; Methods in Molecular Medicine; 1999, HIV Volume 17, I; 35-40; DOI:10.1385/0-89603-369-4:35) 12 μ g, Rp3-S expression plasmid pCDNA3.1-Rp3-S DNA 12 μ g add 62 μ l2M CaCl in above-mentioned dna solution ₂,, pressure-vaccum mixing gently.Get a new 1.5ml centrifuge tube (pipe B), add 500 μ l2xHBS (280mMNaCl, 1.5mMNa ₂HPO ₂, 50mMHepes, pH 7.10); 500 μ l pipe A solution is dropwise added in the pipe B solution, while adding with the rifle head or flick mixing, room temperature placement 20-30min; Above-mentioned 1ml mixing liquid is dropwise added in the 293T Tissue Culture Dish equably, and transfection 8 renews bright substratum 10ml to cell to 12h, behind the transfection 48h; Collect the supernatant of culture medium of 293T cell; 3000 change low-speed centrifugal 5min removes cell debriss, and supernatant removes by filter tiny cell debris and possible bacterium with 0.45 μ m filter again, frozen in-80 ℃ or directly be used for infection experiment with filtering the viral supernatant packing of getting well.

Mouse source monoclonal antibody making processes to the S total length is following: the applicant at first will contain pseudovirus (HIV/Rp3-S) the injection BALB/c mouse of Rp3-S, after then mouse being killed its spleen cell taken out and form with Sp2/0 myeloma cell's fusion) merge.The Hybridoma Cell Culture supernatant carries out the screening of Rp3-S antibody positive with ELISA, and envelope antigen is the Rp3-S1 (embodiment 4) of pseudovirus HIV/Rp3-S and purifying.To select male clone's enlarged culturing and abdominal injection and go into, and collect ascites with the source mouse.It is tired and identifies through ELISA subsequently.For fear of the cross reaction of these monoclonal antibodies and pET32a tag, before using, all monoclonal antibodies all neutralize with the bacterial lysate that contains pET32a tag.

Next use the epitope of monoclonal antibody in the protein hybridization experimental test S protein fragments, the steps include:

With 1 * sample preparation liquid (50mM Tris-HCl (pH6.8); 2% (W/V) SDS, 0.1% (W/V) tetrabromophenol sulfonphthalein, 10% (V/V) glycerine; 1% (W/V) beta-mercaptoethanol) the S fragment albumen of purifying and in boiling water bath, boiling 10 minutes separates with 12% SDS-PAGE then.Then with albumin glue or carry out coomassie brilliant blue staining or forward on the pvdf membrane.Pvdf membrane seals in the TBS that contains 5% (mass volume ratio) skim-milk (pH 7.6 for 20mMTris base, 137mMNaCl) solution and spends the night.With TBST (containing volume ratio is 0.1%Tween-20TBS solution) washing 3 times, each 10min.The S albumen monoclonal antibody of the S protein antibodies that the dna immunization that dilutes with 1:1000 then obtains, 1:250 dilution was hatched one hour for 37 ℃, and antibody incubation liquid is for containing the TBST of 1% (mass volume ratio) skim-milk.Discard an anti-Incubating Solution, with TBST washing 3 times, each 10min.The sheep anti mouse two that adds AP mark 1:2000 dilution resists 37 ℃ and hatched one hour.Discard two anti-Incubating Solutions, with TBST washing 3 times, each 10min.Pvdf membrane shows in 10mL SEAP colour developing liquid (33 μ l5-bromo-4-chloro-indole phosphoric acid salt+66 μ l chlorination nitro blue tetrazoliums) lucifuge, and timely termination reaction.Four all monoclonal antibodies all can be special identification S280-455 rather than pET32a Tag or other four S that block expression.It is important immunodominant region that these data have been confirmed this zone.

SEQUENCE LISTING

< 110>Wuhan Virology Institute,Chinan academy of Sciences

< 120>bat SARS appearance coronavirus spike protein immunity determining area and preparation method and purposes

< 130>bat SARS appearance coronavirus spike protein immunity determining area and preparation method and purposes

<160> 9

<170> PatentIn version 3.1

<210> 1

<211> 176

<212> PRT

< 213>bat SARS appearance coronavirus

<400> 1

Ile Asp Cys Ala Gln Asn Pro Leu Ala Glu Leu Lys Cys Thr Ile Lys

1 5 10 15

Asn Phe Asn Val Ser Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

20 25 30

Ser Pro Thr Gln Glu Val Ile Arg Phe Pro Asn Ile Thr Asn Arg Cys

35 40 45

Pro Phe Asp Lys Val Phe Asn Ala Thr Arg Phe Pro Asn Val Tyr Ala

50 55 60

Trp Glu Arg Thr Lys Ile Ser Asp Cys Val Ala Asp Tyr Thr Val Leu

65 70 75 80

Tyr Asn Ser Thr Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

85 90 95

Ser Lys Leu Ile Asp Leu Cys Phe Thr Ser Val Tyr Ala Asp Thr Phe

100 105 110

Leu Ile Arg Ser Ser Glu Val Arg Gln Val Ala Pro Gly Glu Thr Gly

115 120 125

Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

130 135 140

Val Ile Ala Trp Asn Thr Ala Lys Gln Asp Gln Gly Gln Tyr Tyr Tyr

145 150 155 160

Arg Ser His Arg Lys Thr Lys Leu Lys Pro Phe Glu Arg Asp Leu Ser

165 170 175

<210> 2

<211> 1998

<212> DNA

< 213>bat SARS appearance coronavirus

<400> 2

atgaaaattt taattcttgc tttcctagct agtctagcta aagcacaaga aggatgtggc 60

attatcagtc gaaaacctca gccaaaaatg gcacaagtct cttcttctcg tagaggtgtg 120

tactataatg atgacatttt tcgttctaat gtactacacc tgacgcagga ttatttcctg 180

ccatttgatt caaatttaac acagtacttt tctcttaatg ttgattcaga taggtttacc 240

tactttgaca atcctatttt agactttggt gacggcgtct acttcgctgc tactgaaaag 300

tctaatgtaa ttaggggctg gatttttggt tccactttcg ataacacaac ccagtcagct 360

gttatagtta ataattccac acacattatt atacgtgtgt gcaacttcaa cttatgtaaa 420

gaacctatgt atacagtgtc tcgtggtgca caacaatcat cttgggttta tcagagtgca 480

ttcaattgca catatgatag agtggaaaaa agctttcagc tcgacactgc tcctaaaact 540

ggaaatttta aagacctacg tgagtatgtc tttaagaatc gggatggctt tctcagtgtt 600

taccaaactt atacagctgt taatttacct agaggattac ctattggctt ttcagttttg 660

aggccaattc tcaaactgcc ctttggaatt aacattacat cttatagagt tgttatggct 720

atgtttagcc aaactacttc taatttccta ccagaaagtg ctgcttatta tgttggtaat 780

ttaaaataca ccactttcat gcttagtttt aatgaaaatg ggactattac caatgctatt 840

gattgtgctc aaaacccact tgctgaacta aaatgcacca ttaaaaattt caatgtcagc 900

aagggaatct accaaacatc taacttcaga gtttcgccaa ctcaggaagt tattagattc 960

ccaaacatta caaatcgttg tccttttgac aaagttttta atgctacacg ctttcctaat 1020

gtgtatgcgt gggagagaac taaaatttct gattgtgttg ctgactacac tgttctctac 1080

aactcaactt ctttctcaac ttttaagtgc tatggagttt ctccttctaa gttgattgat 1140

ttatgcttta caagtgtgta tgctgacaca ttcttgataa gatcttcaga agtaagacaa 1200

gttgcaccgg gtgaaactgg tgtcattgct gactataatt acaagctgcc tgatgatttt 1260

actggttgcg taatagcctg gaatactgca aagcaggatc aaggtcagta ttactacagg 1320

tctcaccgga agactaaact taaacctttt gagagagacc tttcttctga tgaaaatggt 1380

gtacgtactc ttagtactta cgacttctac cctagtgtgc cggttgctta tcaggctact 1440

agggtggttg tactgtcatt tgaactacta aacgcacctg caacagtttg tggacctaaa 1500

ttatccacac aacttgttaa gaaccagtgt gtcaatttta attttaatgg actcaaaggt 1560

actggtgttt tgactgaatc atcaaagaga tttcagtcat ttcaacaatt tggtcgtgac 1620

acgtctgatt ttactgactc cgtgcgtgac ccacaaacat tagaaatact tgacatttca 1680

ccatgctctt ttggtggtgt tagtgtaatt acaccaggaa caaatgcttc ttctgaagtg 1740

gctgttcttt atcaagatgt taactgtact gacgtgccag cagcaattca tgcagatcaa 1800

ctaacaccag cttggcgtgt ttattcaact ggaacaaatg ttttccaaac acaggctggc 1860

tgtcttatag gagctgaaca tgttaatgct tcgtatgagt gtgacatccc tattggtgct 1920

ggcatttgtg ctagctacca tacagcttct actttacgta gtgtaggtca gaaatccatt 1980

gtggcttaca ctatgtct 1998

<210> 3

<211> 243

<212> DNA

< 213>bat SARS appearance coronavirus

<400> 3

atgaaaattt taattcttgc tttcctagct agtctagcta aagcacaaga aggatgtggc 60

attatcagtc gaaaacctca gccaaaaatg gcacaagtct cttcttctcg tagaggtgtg 120

tactataatg atgacatttt tcgttctaat gtactacacc tgacgcagga ttatttcctg 180

ccatttgatt caaatttaac acagtacttt tctcttaatg ttgattcaga taggtttacc 240

tac 243

<210> 4

<211> 597

<212> DNA

< 213>bat SARS appearance coronavirus

<400> 4

tttgacaatc ctattttaga ctttggtgac ggcgtctact tcgctgctac tgaaaagtct 60

aatgtaatta ggggctggat ttttggttcc actttcgata acacaaccca gtcagctgtt 120

atagttaata attccacaca cattattata cgtgtgtgca acttcaactt atgtaaagaa 180

cctatgtata cagtgtctcg tggtgcacaa caatcatctt gggtttatca gagtgcattc 240

aattgcacat atgatagagt ggaaaaaagc tttcagctcg acactgctcc taaaactgga 300

aattttaaag acctacgtga gtatgtcttt aagaatcggg atggctttct cagtgtttac 360

caaacttata cagctgttaa tttacctaga ggattaccta ttggcttttc agttttgagg 420

ccaattctca aactgccctt tggaattaac attacatctt atagagttgt tatggctatg 480

tttagccaaa ctacttctaa tttcctacca gaaagtgctg cttattatgt tggtaattta 540

aaatacacca ctttcatgct tagttttaat gaaaatggga ctattaccaa tgctatt 597

<210> 5

<211> 525

<212> DNA

< 213>bat SARS appearance coronavirus

<400> 5

gattgtgctc aaaacccact tgctgaacta aaatgcacca ttaaaaattt caatgtcagc 60

aagggaatct accaaacatc taacttcaga gtttcgccaa ctcaggaagt tattagattc 120

ccaaacatta caaatcgttg tccttttgac aaagttttta atgctacacg ctttcctaat 180

gtgtatgcgt gggagagaac taaaatttct gattgtgttg ctgactacac tgttctctac 240

aactcaactt ctttctcaac ttttaagtgc tatggagttt ctccttctaa gttgattgat 300

ttatgcttta caagtgtgta tgctgacaca ttcttgataa gatcttcaga agtaagacaa 360

gttgcaccgg gtgaaactgg tgtcattgct gactataatt acaagctgcc tgatgatttt 420

actggttgcg taatagcctg gaatactgca aagcaggatc aaggtcagta ttactacagg 480

tctcaccgga agactaaact taaacctttt gagagagacc tttct 525

<210> 6

<211> 438

<212> DNA

< 213>bat SARS appearance coronavirus

<400> 6

actgcaaagc aggatcaagg tcagtattac tacaggtctc accggaagac taaacttaaa 60

ccttttgaga gagacctttc ttctgatgaa aatggtgtac gtactcttag tacttacgac 120

ttctacccta gtgtgccggt tgcttatcag gctactaggg tggttgtact gtcatttgaa 180

ctactaaacg cacctgcaac agtttgtgga cctaaattat ccacacaact tgttaagaac 240

cagtgtgtca attttaattt taatggactc aaaggtactg gtgttttgac tgaatcatca 300

aagagatttc agtcatttca acaatttggt cgtgacacgt ctgattttac tgactccgtg 360

cgtgacccac aaacattaga aatacttgac atttcaccat gctcttttgg tggtgttagt 420

gtaattacac caggaaca 438

<210> 7

<211> 306

<212> DNA

< 213>bat SARS appearance coronavirus

<400> 7

ccatgctctt ttggtggtgt tagtgtaatt acaccaggaa caaatgcttc ttctgaagtg 60

gctgttcttt atcaagatgt taactgtact gacgtgccag cagcaattca tgcagatcaa 120

ctaacaccag cttggcgtgt ttattcaact ggaacaaatg ttttccaaac acaggctggc 180

tgtcttatag gagctgaaca tgttaatgct tcgtatgagt gtgacatccc tattggtgct 240

ggcatttgtg ctagctacca tacagcttct actttacgta gtgtaggtca gaaatccatt 300

gtggct 306

<210> 8

<211> 24

<212> DNA

< 213>bat SARS appearance coronavirus

<400> 8

tgccggatcc attgattgtg ctca 24

<210> 9

<211> 31

<212> DNA

< 213>bat SARS appearance coronavirus

<400> 9

cagtgtcgac ttaagaaagg tctctctcaa a 31

Claims (3)

1. isolating protein, its sequence is the aminoacid sequence shown in the SEQ ID NO:1.

2. the said proteic preparation method of claim 1 the steps include:

A. pcr amplification: with bat SARS appearance coronavirus S gene is template design primer, amplification:

Forward primer: 5 '-TGCCGGATCCATTGATTGTGCTCA-3 ' is SEQ ID NO.8;

Reverse primer: 5 '-CAGTGTCGACTTAAGAAAGGTCTCTCTCAAA-3 ' is SEQ ID NO.9;

The reagent of PCR reaction is following: with the 10xTaq polymerase buffer of 5 μ l, the dNTP mixture of 4 μ l, the TaqDNA polysaccharase of 0.25 μ l, the plasmid pCDNA3.1-Rp-S that 1 μ l contains the Rp3-S gene, primer mixes each sterilized water with 1 μ l and 37.75 μ l;

The PCR reaction conditions: 30 seconds, 72 ℃ of 30 seconds, 56 ℃ renaturation of 300 seconds, 94 ℃ sex change of 94 ℃ of preparatory sex change were extended 60 seconds, and 72 ℃ of last extensions 600 seconds circulates after 30 times;

B, be connected on the expression vector pET32a after cutting with BamHI and XhoI enzyme through the fragment that obtains after the above-mentioned amplification, and order-checking is confirmed errorless;

C, with behind the recombinant plasmid purifying, transform the BL21 competent cell, contain on the amicillin resistance flat board being incubated at; Next day, the picking mono-clonal was cultivated, then final concentration be in the substratum of 0.3mM IPTG 30 ℃ induced 6 hours; Carry out purifying after collecting the thalline ultrasonic disruption, carry out purifying, in SDS-PAGE, detect proteic purity behind the purifying, promptly obtain target protein with His Tag purification kit.

3. the application of the said albumen of claim 1 in identifying mouse anti S monoclonal antibody epi-position.

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