CN1733922A - Super antigen fusion protein and its application method - Google Patents

Abstract

The invention relates to a superantigen fusion protein comprising, E2 Sprotein peptide fragment encoding at least one portion of severe acute respiratory syndrome viruses, and a peptide fragment for conveying and shifting proteins to cytoplasm function from outside the cell, wherein the E2 Sprotein peptide fragment encoding the severe acute respiratory syndrome viruses includes SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, or SEQ ID No.4, The invention also provides the nucleotide sequence for encoding the superantigen fusion protein, which comprises SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, wherein the nucleotide sequence shows corresponding specificity of the protein based on the escherichia coli expression system.

Description

Superantigen fusion rotein and application method thereof

Technical field

The invention relates to a kind of superantigen fusion rotein and application method thereof, refer to that especially a kind of antigen that is engaged in presents cell, effective induce antibody is to suppress the fusion rotein of virus infection and the reaction of blocking-up superantigen.

Background technology

Virus is small, can't independently breed, must parasitize in the cell and can survive, so belong to biological and abiotic between; Coronavirus (Coronaviruses) is a single-stranded RNA virus, with receptor on the target cell in conjunction with after, form a vesica, (endocytosis) enters in the cell via endocytosis, by reverse transcription, can transfer the Yeast Nucleic Acid (RNA) of virus to DNA, embed again in the cell chromosome, utilize host cell to make virus required albumen and genetic material, under the catalysis of ferment, after albumen and the genetic material assembling, in cell, discharge, and destroy host cell.

Virus is assert receptor on the cell surface, for example behind the CD4 and CCR5 antigen on the HIV identification T cell surface, can enter cell; Coronavirus is then assert the amido (Aminopeptidase-N) on the cells such as lungs or kidney, and promptly the structure of CD13 susceptor can be invaded.Therefore with the viral important directions of biotechnology development antagonism, design medicine exactly and stop virus and surface antigen combination, to stop poisoning intrusion: but above-mentioned germ possesses superantigen, can be directly cause fierce inflammatory response with bringing out after susceptor on the T cell combines the T cell to produce a large amount of cytokines (interlukin) or γ-interferon, even allow bonded T cell enter dead path (as the dead apoptosis of formula), if therefore can remove the binding mechanism of superantigen and cell, virus capable of blocking is again invaded by the cell susceptor, then might prevent or alleviate infection symptoms.

In immunity system, all can show one's own T cell receptor (T Cell Receptor on the cytolemma of each T cell, TCR), nearly 1,000,000 sophisticated T cells go on patrol in our health, see through TCR and detect the message that is had on the cytolemma that somatocyte in the health or antigen presents, antigen presents complex body MajorHistocomparibility Complex (MHC) existence that cell has a kind of recognizable extraneous protein, win and just to be presented in surface of cell membrane after peptide (peptide) combines with one section at MHC, and MHC and the formed complex body of victory peptide, be exactly to provide message specially, as the media of identification oneself with nonego to TCR; Yet, the research of endeavouring via scientist, after finding that SARS virus is in intrusive body, certain a part of can combination of sour jujube albumen on its mantle (spike protein) with the TCR on the T cell, and not needing antigen to present the participation of the MHC molecule of cell, think that this normal cell is an external effractor a message by mistake, thereby bring out a large amount of propagation of T cell or produce a large amount of cytokines and disengage immediately, thereby vilify self cell wantonly, cause immunity or inflammatory response; According to above research, can know that SARS sour jujube albumen has the characteristic of superantigen (super antigen) really, but what part is the Amino acid sequence of this section superantigen be located in, need full and accurately inquire into, and have research to infer that the position of this superantigen is to be located in the 680th to 1050 position of SARS sour jujube albumen amido acid sequence recently, but still must further confirm.

Cause autoimmune reaction when preventing the SARS virus invasion, can reach by the system of similar vaccine.And vaccinated key is to make unborn antibody and the viral lymphocyte generation effect of remembering of storage in the final infected pin main body.The present invention finds by the effective induce antibody strategy of fusion rotein, carry out the immunity of superantigen subregion, bring out high-tensile strength valency antibody, make the intravital immunocyte of healthy person produce the antibody that to recognize SARS virus superantigen district, when SARS virus infects, this antibody combines with SARS virus superantigen district, under the situation of not overstimulation T cell proliferation, alleviates and causes superantigen or inflammatory response.The present invention is also by this fusion rotein transmission system, and the antibody that carries out cell susceptor CD13 land brings out, and the antibody that so brings out can be blocked the infringement cell of SARS virus, reaches prevention SARS purpose.

Carry out the research work of SARS, the most important condition must obtain SARS virus earlier exactly, but because this virus is by flying Droplets contagion, therefore will isolate the virus of this hyperinfection, must just can carrying out in well-appointed laboratory; Even there has been scientist that the genome sequencing of SARS wild virus strain is finished, external synthetic a large amount of viruses still must rely on specific host system, the restriction that has it to utilize; The present invention is then according to the gene order of the SARS wild virus strain of networking bulletin, translation Amino acid sequence, again with the Amino acid sequence, in general escherichia coli host system, password (Usage codon) upgrading of full blast performance, and by continuous P CR step, synthetic gene sequence can effectively show SARS protein in the escherichia coli host system voluntarily.This kind need virogene entity sample, just can obtain SARS antigen, will more help the research of SARS virus.

Summary of the invention

The object of the present invention is to provide a kind of superantigen fusion rotein and application method thereof, superantigen fusion rotein coding wherein has the E2 sour jujube albumen (E2spike protein) of one section SARS (Severe Acute Respiratory Syndrome) virus to win too fragment, and one section victory with displacement (translocation) function fragment too, simultaneously, for in the virus that must not obtain hyperinfection, still can be in the sour jujube albumen of external synthesis of severely acute respiratory syndrome virus E2, the present invention comprises that also a kind of coding has the nucleotide sequence of superantigen fusion rotein, and the superantigen fusion rotein that is derived from virus can be showed in the escherichia coli host system.

Another object of the present invention is to provide a kind of superantigen Sheng Tai district of the T of being engaged in cell capable of blocking, particularly with the E2 sour jujube albumen of SARS (Severe Acute Respiratory Syndrome) virus be target victory too, utilize the antibodies mode, engage with E2 sour jujube albumen superantigen zone in advance, and avoid causing excessive immune response and anaphylaxis.

Another object of the present invention is to provide the joint Sheng Tai district of CD13 receptor on a kind of T of being engaged in cell capable of blocking, particularly with the E2 sour jujube albumen of SARS (Severe Acute Respiratory Syndrome) virus be target victory too, utilize the antibodies mode, blocking virus engages with CD13 receptor on the immune t-cell, engage with E2 sour jujube albumen superantigen zone in advance, and avoided SARS (Severe Acute Respiratory Syndrome) virus to engage the back and invade host cell via immune t-cell CD13.

Whether the present invention can be used for detecting the SARS (Severe Acute Respiratory Syndrome) virus infection, and because the antibody that is brought out can be avoided the virus infection host cell, and can reduce because immune t-cell engages excessive immune response and the anaphylaxis that the back is caused with SARS (Severe Acute Respiratory Syndrome) virus, so fusion rotein of the present invention also can be used for alleviating or treating the infection symptoms of SARS (Severe Acute Respiratory Syndrome) virus.

For achieving the above object, the present invention has disclosed two kinds of superantigen fusion roteins, is to encode too fragment of the identical victory of E2 sour jujube albumen (the E2 spike protein) gene of at least a portion SARS (Severe Acute Respiratory Syndrome) virus is arranged; And one section victory fragment too with cell in conjunction with (binding) and displacement (translocation) function; Wherein, this SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen wins too that fragment comprises SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

The present invention also comprises that a kind of coding has the nucleotide sequence of superantigen fusion rotein, comprising: SEQ IDNO.5; SEQ ID NO.6; SEQ ID NO.7; And SEQ ID NO.8; Wherein, these nucleotide sequences are to show its corresponding specificity protein with the intestinal bacteria representation system.

The victory that the present invention also comprises a kind of T of being engaged in cell too, this victory comprises very much the sour jujube albumen of at least a portion SARS (Severe Acute Respiratory Syndrome) virus E2, this victory comprises very much SEQ ID NO.1, SEQID NO.2, SEQ ID NO.3 and SEQ ID NO.4.

The present invention also comprises a kind of victory medical composition too that contains in order to be engaged in the T cell, and comprising coding has the SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen of at least a portion to win too fragment; Wherein, this SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen wins too that fragment comprises SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4.

The present invention wins sequence too can comprise also that one section has the nucleotide fragments of displacement (translocation) function or wins too, and this shift sequence kind is not limit, major function is to bring very much cell interior in the victory that will be connected, first and the two section functional part that is preferably with Rhodopseudomonas exotoxin A (pseudomonas exotoxin A) (sees also United States Patent (USP) 5 very much as engaging with the displacement victory, 705,163).

Medical component of the present invention also comprises and contains SEQ ID NO.1, and the victory of SEQ ID NO.2 or its mixture too can be in order to as the vaccine that bring out passive immunization; And containing SEQ ID NO.3, the victory of SEQ IDNO.4 or its mixture too then can be in order to the vaccine as manufacturing SARS (Severe Acute Respiratory Syndrome) virus.

Superantigen fusion rotein of the present invention comprises four sections E2 sour jujube albumen (E2spike protein) fragments from SARS (Severe Acute Respiratory Syndrome) virus, the SARS E2 sour jujube protein sequence (NP 828851) that finds in its Amino acid sequence and the U.S. state-run biotechnology data center (NCBI) database is identical, but for SARS E2 sour jujube albumen can be synthesized by external, it is the method that 92126644 Taiwan patent is disclosed that the present invention utilizes application number, to change nucleotide sequence, but do not change its Amino acid of encoding out and carry out the synthetic of SARS E2 sour jujube protein part sequence, the nucleotide sequence behind the upgrading comprises: SEQ IDNO.5:SEQ ID NO.6; SEQ ID NO.7 and SEQ ID NO.8.

Description of drawings

Fig. 1 is the plastid collection of illustrative plates of the embodiment of the invention one; 1A is pET-PE-SA; 1B is pET-PE-SB; 1C is pET-PE-SC; 1D is pET-PE-SD;

Fig. 2 is the embodiment of the invention three ferment immunosorption spotting methods, and the reaction result that different time-histories are cultivated: A1 is the result of fresh peripheral blood monocyte and PE-SA reaction; A2 is fresh peripheral blood monocyte and the result of PE-SA reaction after 7 days; A3 is fresh peripheral blood monocyte and the result of PE-SA reaction after 14 days; B1 is the result of fresh peripheral blood monocyte and PE-SB reaction; B2 is fresh peripheral blood monocyte and the result of PE-SB reaction after 7 days; B3 is fresh peripheral blood monocyte and the result of PE-SB reaction after 14 days; C1 is the result of fresh peripheral blood monocyte and PE-SC reaction; C2 is fresh peripheral blood monocyte and the result of PE-SC reaction after 7 days; C3 is fresh peripheral blood monocyte and the result of PE-SC reaction after 14 days; D1 is the result of fresh peripheral blood monocyte and PE-SD reaction; D2 is fresh peripheral blood monocyte and the result of PE-SD reaction after 7 days; D3 is fresh peripheral blood monocyte and the result of PE-SD reaction after 14 days;

Fig. 3 is the embodiment of the invention three a ferment immunosorption spotting methods countings bar chart, wherein has respectively to contain the PE displacement and win and win too four kinds of fusion roteins of SA, SB, SC and SD with four sections very much, and only contains the PE displacement and win too albumen;

Fig. 4 is the detection of the embodiment of the invention four antibody power valencys, and wherein A, B, C and D four figure represent respectively and contain PE displacement and win and win the too fusion rotein of SA, SB, SC and SD with four sections very much; The solid rectangular fusion rotein of the present invention that is; Hollow rectangular for only containing PE displacement victory albumen too.

Embodiment

For more understanding technology contents of the present invention, be described as follows especially exemplified by preferred embodiment.

Embodiment one, SARS E2 sour jujube protein part win too synthesizing of sequence

In the U.S. state-run biotechnology data center (NCBI) database, find SARS E2 sour jujube protein sequence (accession number:NP 828851, SEQ.ID.NO.9), get that 680-1050 Amino acid is divided into four fragments on the sequence, carry out Nucleotide upgrading and segmental synthetic amplification, as the external synthetic target of the embodiment of the invention.

It is the method that 92126644 Taiwan patent is disclosed that present embodiment can utilize application number, and the proteic heterologous protein of viral sour jujube can be showed by the intestinal bacteria system, with efficient carry out external synthetic; The emphasis of upgrading is mainly at the nucleotide fragments with the wild virus strain, not influencing the Amino acid that it shows originally, and under the situation that can effectively show, carries out the change of single Nucleotide in the escherichia coli host system.

Utilize commercially available intestinal bacteria plastid pET230, as polymerase chain reaction (polymerasechain reaction, PCR) masterplate (template) of amplification fragment nucleic acid.4 sections nucleotide fragments of present embodiment synthetic are respectively SA, SB, SC and SD and win too, have utilized totally 29 pairs of introductions to carry out fragment and have amplified, and the right sequence of all introductions is asked for an interview table 1; Wherein this forward introduction (forward, code name F1) is the partial sequence homology with intestinal bacteria plastid pET230.

Table 1, introduction are to sequence table

Win too code name	The sequence code name	Base sequence
			SA	SA-F1	5′-CCC TCA GAA TTC GAG AAC ACC ATC GCT ATC CCG A-3′
SA-R1	5′-GAT GGA GAT GGA GAA GTT GGT CG G GAT AGC GAT GGT GTT CTC GAG TGC TGA GGG-3′
		SA-R2		5′-GGA GGT TTT AGC CAT GGA AAC CGG CAT AAC TTC GGT GGT GAT GGA GAT GGA GAA-3′
SA-R3	5′-TTC GGT GGA GTC ACC GCA GAT GTA CAT GTT GCA GTC AAC GGA GGT TTT AGC CAT-3′
		SA-R4		5′-GGT GCA GAA GGA ACC GTA CTG CAG CAG CAG GTT AGC GCA TTC GGT GGA GTC ACC-3′
SA-R5	5′-AGC GAT ACC GGA CAG AGC ACG GTT CAG CTG GGT GCA GAA GGA ACC-3′
		SA-R6		5′-TTT TGA ATT CAC GGG TGT TAC GGT CCT GTT CAG CAG CGA TAC CGG ACA G-3′
SB	SB-F1				5′-CCC TCA GAA TTC GAG GTT TTC GCT CAG GTT AAA-3′
		SB-R1	5′-GGT CGG GGT TTT GTA CAT CTG TTT AAC CTG AGC GAA AAC CTC GAG TGC TGA GGG-3′
	SB-R2			5′-CAG GAT CTG GGA GAA GTT GAA ACC ACC GAA GTA TTT CAG GGT CGG GGT TTT GTA-3′
		SB-R3	5′-TTC GAT GAA GGA ACG TTT GGT CGG TTT CAG CGG GTC CGG CAG GAT CTG GGA GAA-3′
	SB-R4			5′-ACC AGC GTC AGC CAG GGT AAC TTT GTT GAA CAG CAG GTC TTC GAT GAA GGA ACG-3′
		SB-R5	5′-GTT GAT GTC ACC CAG GCA TTC ACC GTA CTG TTT CAT GAA ACC AGC GTC AGC CAG-3′
	SB-R6			5′-CAG ACC GTT GAA TTT CTG AGC GCA GAT CAG GTC ACG AGC GTT GAT GTC ACC CAG-3′
		SB-R7	5′-CAT GTC GTC GGT CAG CAG CGG CGG CAG AAC GGT CAG ACC GTT GAA TTT-3′
	SB-R8			5′-TTT TGA ATT CCA GAG CAG CGG TGT AAG CAG CGA TCA TGT CGT CGG TCAG-3′

SC	SC-F1	5′-CCC TCA GAA TTC GAG GTT TCC GGT ACC GCT ACC GCT-3′
			SC-R1	5′-AGC ACC GAA GGT CCA ACC AGC GGT AGC GGT ACC GGA AAC CTC GAG TGC TGA GGG-3′
	SC-R2	5′-AGC CAT CTG CAT AGC GAA CGG GAT CTG CAG AGC AGC ACC AGC ACC GAA GGT CCA-3′
			SC-R3	5′-CAG AAC GTT CTG GGT AAC ACC GAT ACC GTT GAA ACG GTA AGC CAT CTG CAT AGC-3′
	SC-R4	5′-TTT GTT GAA CTG GTT AGC GAT CTG TTT CTG GTT TTC GTA CAG AAC GTT CTG GGT-3′
			SC-R5	5′-GGA GGT GGT GGT CAG GGA TTC CTG GAT CTG GGA GAT AGC TTT GTT GAA CTG GTT-3′
	SC-R6	5′-AGC GTT CTG GTT AAC AAC GTC CTG CAG TTT ACC CAG AGC GGA GGT GGT GGT CAG-3′
			SC-R7	5′-TTT TGA ATT CGG ACA GCT GTT TAA CCA GGG TGT TCA GAG CCT GAG CGT TCT GGT TAAC-3′
	SD	SD-F1			5′-CCC TCA GAA TTC GAG TCC AAC TTC GGT GCT ATC TCC T-3′
SD-R1			5′-GAT GTC GTT CAG AAC GGA GGA GAT AGC ACC GAA GTT GGA CTC GAG TGC TGA GGG-3′
		SD-R2		5′-GAT CTG AAC TTC AGC TTC AAC TTT GTC CAG ACG GGA CAG GAT GTC GTT CAG AAC-3′
SD-R3			5′-GGT CTG CAG GGA CTG CAG ACG ACC GGT GAT CAG ACG GTC GAT CTG AAC TTC AGC-3′
		SD-R4		5′-ACG GAT TTC AGC AGC ACG GAT CAG CTG CTG GGT AAC GTA GGT CTG CAG GGA CTG-3′
SD-R5			5′-GCA TTC GGA CAT TTT GGT AGC AGC CAG GTT AGC GGA AGC ACG GAT TTC AGC AGC-3′
		SD-R6		5′-TTT ACC GCA GAA GTC AAC ACG TTT GGA CTG ACC CAG AAC GCA TTC GGA CAT TTT-3′
SD-R7			5′-GTG CGG AGC AGC CTG CGG GAA GGA CAT CAG GTG GTA ACC TTT ACC GCA GAA GTC-3′
		SD-R8		5′-TTT TGA ATT CAA CGT AGG TAA CGT GCA GGA AAA CAA CAC CGT GCG GAG CAG CCT G-3′

Four sections victorys too respectively with the different reverse introduction of same forward introduction (SA-F1, SB-F1, SC-F1 and SD-F1) collocation (SA-R1～R6, SB-R1～R8, SC-R1～R7 and SD-R1～R8) carry out (SA6 time, SB8 time, SC7 time, SD8 time) PCR for several times, the time conditions that it adopted is: first circulates be 95 ℃-5 minutes; Second circulation be 94 ℃-1 minute, 55 ℃-0.5 minute, 72 ℃-1 minute, carry out 20 altogether and repeat; The 3rd the circulation be 72 ℃-1 minute.

On behalf of the band of product, the PCR product of finishing amplification will on the electrophoresis cut down after with electrophoresis observation product size, and the collection of carrying out product goes out (elution).

Embodiment two, plastid merge

Four sections of synthetic of embodiment are won too SA, SB, SC and SD (please refer to Liao C.W.et al. with the plastid that plastid pET-PE--has Rhodopseudomonas exotoxin A combined function position (binding domain) and a shift function position (translocation domain) respectively, AppliedMicrobiol Biotechnol 143:498-507,1995), utilize the restriction enzyme position of reserving to carry out the contraposition combination, finish four kinds of fusion plastids and be respectively pET-PE-SA, pET-PE-SB, pET-PE-SC and pET-PE-SD (Fig. 1).

Embodiment three, protein performance and purifying

According to the method (Sambrook et al., 1989) of Sambrook, finish synthetic pET-PE-SA, pET-PE-SB, pET-PE-SC and pET-PE-SD with four sections respectively and merge plastid, utilize the performance of IPTG induced protein.

Select for use E.coli BL21 (DE3) pLysS to carry out the protein performance of (winning too).At first with spawn culture in the 100ml LB nutrient solution that contains 200ug/ml ampicilin, be cultured to bacterial concentration and reach OD550 till 0.3; Now adds IPTG in bacterium liquid (USA) 1mM continues to cultivate after 90 minutes, and the cell that is grown is carried out centrifugal collection for isopropylthio-β-D-galactoside, Promege; Mode with the operation of freeze-thaw repetitiousness makes the membrane structure of the cell that has target protein slightly loose, add lysate 10ml and (contain the 0.9mg/ml N,O-Diacetylmuramidase this moment, the DNaseI of 1mlPMSF and 0.064mg/ml), placed room temperature following 10 minutes, now adds 1ml10%Triton X-100 again, place room temperature following 10 minutes, and collected protein in centrifugal 10 minutes with 12000xg afterwards; Clean with urea respectively again with 1M and 2M; At last, collected protein inclusion body (Inclusion body) is dissolved among the 8M urea of 4ml.

Now is again with commercially available pET His-Tag purification system (Novagen, USA), carry out following experiment according to the description of test book: with the cell mass collected method by the ultrasound concussion, molten loosing in the ice-cold binding buffer of 4ml (containing 5mM imidazole-0.5M, Nacl-20mM and Tris-HClpH7.9) scattered up to agglomerate; Now carries out in 4 ℃ from, centrifugal 15 minutes of 1200xg, the supernatant liquor after centrifugal is poured into (tubing string is His-Bind metal chelation resinimmobilized with Ni in the tubing string again ²⁺), it is last that (contain 0.5M imidazole, 0.5m NaCl and 20mM Tris-HCl pH7.9) sweep away collection with a damping fluid with the protein of gluing in tubing string again.

Utilization is finished four kinds of fusions four kinds of fusion rotein: PE-SA, PE-SB, PE-SC and PE-SD that plastid showed with quadrat method.

Embodiment three, cellular immunization experiment

A. the monocytic separation of peripheral blood

After obtaining the blood of normal adults, with 1: 1 mixed in Hanks ' balanced salt solution (Hanks, balanced salt solution kit, Life Technologies, Rockville, MD).

According to the method for Sacerdote (Sacerdote, 1991#1979) and Ficoll-Paque solution (Amersham Biosciences, Uppsala Sweden) isolate the peripheral blood monocyte.The Ficoll-Paque solution that is mixed with blood is carried out centrifugal (600xg, 30 minutes), to have monocytic solution afterwards and be moved to another centrifuge tube, after Hahks ' balanced salt solution cleaning 2 times, under 1200xg centrifugal 10 minutes again, promptly finish the monocytic separation of peripheral blood.

Utilize Gong method (Gong, 2000#1976), with immature peripheral blood monocyte differentiate dendritic cell (dendritic cells, DCs).At first the peripheral blood monocyte is scattered in the cell culture fluid, and cell is attached in the culture plate; Cultivated 2 hours down in 37 ℃; Now removes the cell that does not attach; (VA) nutrient solution and GM-CSF (800IU/ml) and IL-4 (500U/ml) cultivated the cell that attaches 6 days together for Mediatech, Herndon to utilize the RPMI 1640 contain 1% human serum again; Now is in the 0th day, adds cytokine (cytokines) in the 2nd day or the 4th day respectively; Whether in the time of the 6th day, collecting the cell that does not attach has the molecule marker that belongs to dendritic cell to analyze.

The FACScan buffer solution for cleaning that dendritic cell utilization under collecting is contained PBS, 0.2%FBS and 0.5% trisazo-sodium (sodium azide); Now adds the anti-mankind's of FITC-or PE-bond antibody (CD1a, CD3, CD56, CD80, HLA-A, B, C and HLA-DR; PharMingen, San Diego, CA), and in acting on 30 minutes on ice, again with the FACScan buffer solution for cleaning.Utilize flow cytometer (FACScalibur flow cytometer, BectonDickinson, Mountain View, CA) and " CellQuest " software carries out data analysis.

B.IFN-γ ferment immunosorption spotting method (ELISPOT Assay)

According to people such as Miyahira (Miyahira, 1995#677) and people (Murali-Krishna such as Murali-Krishna, document 1998#698) carries out the part of ferment immunosorption spotting method experiment and revises, the single detection at the narrow spectrum CD8+T cell of antigen SA, SB, SC and SD tool.

(Millipore, Bedford are covered with antibody (the 10 μ g/ml that one deck contains anti-human INF-γ on MA) at 96 hole filter discs; PharMingen) PBS (50 μ l); Cultivate overnight down in 4 ℃; Now washes culture hole, and is overlying in the hole with the nutrient solution resistance that contains 10% foetal calf serum; Prepare the fresh of different concns or the peripheral blood mononuclear cell of cultivating in addition, with the dendritic cell (as embodiment three As described in) of 10: 1 mixed differentiation, with 1 * 10 from autogenous cell ⁶Concentration adds in the culture hole after resistance is covered, wherein employed dendritic cell be not through or through four kinds of fusion rotein-PE-SA, PE-SB, PE-SC and 2 hours cell of PE-SD processing.

After cultivation, culture hole is once more through cleaning, and adding contains antibody (the 5 μ g/ml that bond has the anti-human INF-γ of mouse of vitamin H; PharMingen) behind the PBS (50 μ l), cultivate overnight down in 4 ℃; After now cleans porose disc 6 times, add and contain 1.2 μ g/ml avidin alkaline phosphatases (avidin-alkaline phosphatase, Sigma, St.Louis, 50 μ l PBS MO), and in room temperature, cultivating 2 hours; Now adds 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium solution (the Boehringer Mannheim of 50 μ l again, Indianapolis, IN, USA), cultivate after 20 minutes under the room temperature, promptly can be observed the generation of spot; Remove afterwards and be subjected to matter solution, clean culture hole with tap water again, get final product termination reaction; With after the culture plate drying, calculate color spot quantity at last with dissecting microscope.

Ferment immunosorption spotting method is in order to detect the quantity at PE-SA, PE-SB, PE-SC and four kinds of CD 8+T cells that fusion rotein reflected of PE-SD; Fig. 2 is the result of ferment immunosorption spotting method, can observe the reaction result that different time-histories are cultivated from figure; Spot counting back is represented as Fig. 3 with bar chart, can obviously find out behind mouse immune the 7th day by figure, in the mice serum of injection PE-SA, PE-SC and three kinds of fusion roteins of PE-SD, the generation of INF-γ improves, arrived behind the mouse immune the 14th day, the PE-SC fusion rotein will bring out serious immune response, and make the INF-γ that a large amount takes place in the mouse body.

Embodiment four, animal immune experiment

A. the preparation of animal

6-8 week, big female mice C57BL/6J was available from Univ Nat Taiwan (Taibei, Taiwan), and raised the animal center of setting up hospital in Univ Nat Taiwan.

B. the immunity of animal

Preparation is injected mouse with the amount of 100 μ g respectively from four kinds of fusion rotein-PE-SA, PE-SB, PE-SC and PE-SD of embodiment two, carry out immunoreactive test, prepare simultaneously a part of mouse in addition, injection only contains the protein of Rhodopseudomonas exotoxin A, as the control group; The injection time-histories is for testing the 0th day, the 14th day and the 28th day that begins to carry out.

C. specificity detection of antibodies

Utilize Chengc method (Cheng, 2001#1486)--(Enzyme-linked Immunoabsorbent Assay ELISA), carries out PE-SA, PE-SB in the serum, PE-SC and PE-SD specificity detection of antibodies to the ferment immunoabsorption.

On 96 porose discs, be covered with one deck PE-SA, PE-SB, PE-SC and PE-SD (5 μ g) fusion rotein, cultivate overnight down in 4 ℃; Be overlying in the culture hole with the PBS resistance that contains 20% foetal calf serum again; Obtain serum in carrying out the mouse of immunity after 14 days, utilize PBS to carry out serial dilution, now adds in the culture hole, cultivates 2 hours in 37 ℃; After cleaning culture hole with the PBS that contains 0.05%Tween 20 again, the bond that added 1: 2000 has the anti-ageing mouse IgG of rabbit antibody (the peroxidase-conjugated rabbit anti-mouse IgG antibody of peroxidase, Zymed, SanFrancisco, CA) in culture hole, under room temperature, cultivated 1 hour again; Wash culture plate afterwards, and (Pierce, Rockford IL) launch color, last H with 1M to add 1-Step Turbo TMB-ELISA ₂SO ₄Termination reaction; Utilize the ELISA interpretoscope under the absorption spectrum of 450nm, to carry out result's interpretation.

Fig. 4 is serial dilution serum 1: 100,1: 500 and 1: 1000, the detection of its specificity antibody power valency, the serum of PE-SC and PE-SD fusion rotein mouse is squeezed in result's demonstration, detecting the CD 8+T cell value that is produced exceeds in the amount of squeezing into PE-SA and PE-SB fusion rotein mouse serum (P＜0.01, oneway ANOVA).Found that thus PE-SC and PE-SD fusion rotein itself induce the higher antibody response of animal body, the recipient who shows PE-SC and PE-SD fusion rotein can be subjected to the antibody effect that taken place clinically, and has the effect of protecting the recipient.

Fusion rotein of the present invention can successfully bring out the antibody producing of animal body, because PE-SC and PE-SD fusion rotein can bring out a large amount of generations of INF-γ, SC and the Tai Ji of SD victory may be the superantigen position of SARS (Severe Acute Respiratory Syndrome) virus in the demonstration embodiment of the invention, therefore may cause the animal body of injection PE-SC and PE-SD fusion rotein induce antibody to produce excessive immune response, and the anxiety of life threatening is arranged; But from another viewpoint, because the mass production of antibody is arranged, can use PE-SC and PE-SD fusion rotein future and bring out the specificity antibody that animal body produces and carry out the production of vaccine, further treat the infection of SARS (Severe Acute Respiratory Syndrome) virus.

In addition, two kinds of fusion roteins of PE-SA of the present invention and PE-SB are not because can bring out the excessive immune response of animal body, therefore, can develop into the mechanism that the animal of PI SARS (Severe Acute Respiratory Syndrome) virus is formed passive immunization, as long as injection PE-SA and two kinds of fusion roteins of PE-SB, then can block susceptor CD13 bonded position on the sour jujube albumen of SARS (Severe Acute Respiratory Syndrome) viruses molecule and the T cell in animal body, and the infection of prevention SARS (Severe Acute Respiratory Syndrome) virus.

The foregoing description only is to give an example for convenience of description, and the interest field that the present invention advocated should be as the criterion so that claim is described certainly, but not only limits to the foregoing description.

Sequence table

＜110〉the precious Cord blood of life bank

＜120〉superantigen fusion rotein and application method thereof

<130>P7133/0357

<160>8

<170>PatentIn version 3.3

<210>1

<211>71

<212>PRT

＜213〉SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen

<300>

<308>NP_828851

<309>2004-04-09

<313>(1)..(71)

<400>1

Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile Thr Thr Glu

1 5 10 15

Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys Asn Met Tyr

20 25 30

Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu Gln Tyr Gly

35 40 45

Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile Ala Ala Glu

50 55 60

Gln Asp Arg Asn Thr Arg Glu

65 70

<210>2

<211>97

<212>PRT

＜213〉SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen

<300>

<308>NP_828851

<309>2004-04-09

<313>(1)..(97)

<400>2

Val Phe Ala Gln Val Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr

1 5 10 15

Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro

20 25 30

Thr Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu

35 40 45

Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile

50 55 60

Asn Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val

65 70 75 80

Leu Pro Pro Leu Leu Thr Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala

85 90 95

Leu

<210>3

<211>90

<212>PRT

＜213〉SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen

<300>

<308>NP_828851

<309>2004-04-09

<313>(1)..(90)

<400>3

Val Ser Gly Thr Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala

1 5 10 15

Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile

20 25 30

Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn

35 40 45

Gln Phe Asn Lys Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr

50 55 60

Ser Thr Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln

65 70 75 80

Ala Leu Asn Thr Leu Val Lys Gln Leu Ser

85 90

<210>4

<211>101

<212>PRT

＜213〉SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen

<300>

<308>NP_828851

<309>2004-04-09

<313>(1)..(101)

<400>4

Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg

1 5 10 15

Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly

20 25 30

Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala

35 40 45

Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu

50 55 60

Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr

65 70 75 80

His Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val Phe Leu

85 90 95

His Val Thr Tyr Val

100

<210>5

<211>219

<212>DNA

＜213〉synthetic

<220>

＜223〉coding has can show the proteic nuclear sequence of SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube in the intestinal bacteria representation system

<400>5

aacaccatcg ctatcccgac caacttctcc atctccatca ccaccgaagt tatgccggtt 60

tccatggcta aaacctccgt tgactgcaac atgtacatct gcggtgactc caccgaatgc 120

gctaacctgc tgctgcagta cggttccttc tgcacccagc tgaaccgtgc tctgtccggt 180

atcgctgctg aacaggaccg taacacccgt gaataatag 219

<210>6

<211>297

<212>DNA

＜213〉synthetic

<220>

＜223〉coding has can show the proteic nuclear sequence of SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube in the intestinal bacteria representation system.

<400>6

gttttcgctc aggttaaaca gatgtacaaa accccgaccc tgaaatactt cggtggtttc 60

aacttctccc agatcctgcc ggacccgctg aaaccgacca aacgttcctt catcgaagac 120

ctgctgttca acaaagttac cctggctgac gctggtttca tgaaacagta cggtgaatgc 180

ctgggtgaca tcaacgctcg tgacctgatc tgcgctcaga aattcaacgg tctgaccgtt 240

ctgccgccgc tgctgaccga cgacatgatc gctgcttaca ccgctgctct gtaatag 297

<210>7

<211>276

<212>DNA

＜213〉synthetic

<220>

＜223〉coding has can show the proteic nuclear sequence of SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube in the intestinal bacteria representation system

<400>7

gtttccggta ccgctaccgc tggttggacc ttcggtgctg gtgctgctct gcagatcccg 60

ttcgctatgc agatggctta ccgtttcaac ggtatcggtg ttacccagaa cgttctgtac 120

gaaaaccaga aacagatcgc taaccagttc aacaaagcta tctcccagat ccaggaatcc 180

ctgaccacca cctccaccgc tctgggtaaa ctgcaggacg ttgttaacca gaacgctcag 240

gctctgaaca ccctggttaa acagctgtcc taatag 276

<210>8

<211>309

<212>DNA

＜213〉synthetic

<220>

＜223〉coding has can show the proteic nuclear sequence of SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube in the intestinal bacteria representation system.

<400>8

tccaacttcg gtgctatctc ctccgttctg aacgacatcc tgtcccgtct ggacaaagtt 60

gaagctgaag ttcagatcga ccgtctgatc accggtcgtc tgcagtccct gcagacctac 120

gttacccagc agctgatccg tgctgctgaa atccgtgctt ccgctaacct ggctgctacc 180

aaaatgtccg aatgcgttct gggtcagtcc aaacgtgttg acttctgcgg taaaggttac 240

cacctgatgt ccttcccgca ggctgctccg cacggtgttg ttttcctgca cgttacctac 300

gtttaatag 309

Claims (16)

1. superantigen fusion rotein comprises:

Coding has the E2 sour jujube albumen (E2 spikeprotein) of at least a portion SARS (Severe Acute Respiratory Syndrome) virus to win too fragment;

And one section victory with displacement (translocation) function fragment too;

Wherein, this SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen wins too that fragment comprises SEQ IDNO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4.

2. fusion rotein as claimed in claim 1 is characterized in that, this displacement wins too that fragment is Rhodopseudomonas extracellular toxin (pseudomonas exotoxin).

3. a nucleotide sequence is in the intestinal bacteria representation system, and the E2 sour jujube albumen (E2 spike protein) of narrow spectrum SARS (Severe Acute Respiratory Syndrome) virus is provided in performance, has following sequence:

SEQ ID NO.5；

SEQ ID NO.6；

SEQ ID NO.7; Or

SEQ ID NO.8。

4. nucleotide sequence as claimed in claim 3 is characterized in that, these nucleotide sequences also include too fragment of one section victory with displacement (translocation) function.

5. nucleotide sequence as claimed in claim 4 is characterized in that, this displacement wins too that fragment is the Rhodopseudomonas extracellular toxin.

6. a victory that is engaged in the T cell comprises the E2 sour jujube albumen of at least a portion SARS (Severe Acute Respiratory Syndrome) virus very much, and this victory is very much:

SEQ ID NO.1；

SEQ ID NO.2；

SEQ ID NO.3; Or

SEQ ID NO.4。

7. victory as claimed in claim 6 is characterized in that very much these victorys are produced by an intestinal bacteria representation system.

8. victory as claimed in claim 6 is characterized in that very much, also comprises one section victory with shift function too.

9. victory as claimed in claim 8 is characterized in that very much it is very much the Rhodopseudomonas extracellular toxin that this displacement wins.

10. victory medical composition too that contains in order to be engaged in the T cell, comprising coding has the SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen of at least a portion to win too fragment;

Wherein, this SARS (Severe Acute Respiratory Syndrome) virus E2 sour jujube albumen wins too that fragment comprises SEQ IDNO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

11. medical composition as claimed in claim 10 is characterized in that, also comprises one section victory with shift function too.

12. medical composition as claimed in claim 11 is characterized in that, this victory with shift function is very much the Rhodopseudomonas exotoxin A.

13. medical composition as claimed in claim 10 is characterized in that, is used for the treatment of the infection of serious acute respiratory, road syndrome virus.

14. medical composition as claimed in claim 10, it also comprises pharmaceutically acceptable carrier.

15. medical composition as claimed in claim 10 is characterized in that, comprises this SEQ IDNO.1, the victory of SEQ ID NO.2 or its mixture is brought out the vaccine of passive immunization very much in order to conduct.

16. medical composition as claimed in claim 10 is characterized in that, comprises this SEQ IDNO.3, the victory of SEQ ID NO.4 or its mixture is made the vaccine of SARS (Severe Acute Respiratory Syndrome) virus very much in order to the conduct system.

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