CN1772921A - Bioinformatic screening process of simulated epitope of pathogenic microbe - Google Patents

Abstract

The bioinformatic screening process of simulated epitope of pathogenic microbe includes the first BLAST comparison of LCDV-1 genome sequence to screen out protective antigen protein candidate gene TK and ORF29; the subsequent hydrophobicity, hydrophilicity, antigenicity and transmembrane structure analysis of these two gene expressed proteins and synthesizing 12 kinds of structural analysis results; and final presumption of unknown LCDV-cn corresponding simulated epitopes with I>0 sequences based on the comprehensive antigen index formula. The obtained simulated epitopes are further artificially synthesized and the immunoreactivity of various simulated epitopes are inspected through competitive enzyme-linked immune analysis. The present invention makes best utilization of rich nucleic acid and protein sequence resource to capture the simulated epitope of unknown pathogenic microbe antigen protein through specific bioinformatic analysis.

Description

The information biology screening method of pathogenic micro-organism mimic epitopes

Technical field

The present invention relates to bioinformatic analysis, with the protective antigen mimic epitopes of quick acquisition unknown gene group or antigenicity information pathogeny microorganism, i.e. the information biology screening method of pathogenic micro-organism mimic epitopes to known/genome sequence.

Background technology

The health that appears as human and aquaculture organism of vaccine has been made huge contribution.Along with development of science and technology, the state of the art of vaccine development is also improving constantly.Fa Ming inactivated vaccine was still used widely up to today the earliest; the defective of its maximum is the immunne response imperfection that excites; be difficult to provide effective immanoprotection action, and there is more serious safety issue in attenuated vaccine, has restricted its application to a great extent.When this situation, the nucleic acid vaccine development technology in third generation subunit vaccine and the 4th generation is arisen at the historic moment, and the latter especially is because of it can induce comprehensive body fluid and immunne response, it is all quite convenient to add production preparation and storage, therefore becomes the focus of current vaccine development.

No matter be subunit vaccine or nucleic acid vaccine, its core all is can the antigen protein of mediate protection immunological effect and defining of antigenic determinant.Current antigen protein that adopts and antigenic determinant screening method mainly can be divided into two big classes.One class is the cultivation by the pathogeny microorganism, collects protein purification, passes through the mensuration of a series of bioanalysis until aminoacid sequence then, finally checks its immunogenicity and immunoreactivity with the form of recombinant protein by clonal expression.Its two, utilize phage display peptide library or protein biochip technology, set up thousands of and even ten thousand overlapping peptide storehouse according to the proteinic sequence of known antigens, eluriate the final required mimic epitopes of isolation identification by number wheel is biological.Aforesaid method all exists time-consuming, tediously long and manpower and materials expend huge shortcoming; Simultaneously need a large amount of special reagents and instrument in qualification process, be difficult to accept for most laboratories, this also is a current pathogeny microbial antigen protein analysis progress major cause slowly.

Yet the breakthrough of genomics research provides possibility for the fast development of oppositely immunogenetics research.Gene, gene order-checking have all become breadboard routine techniques at present, and be quick, easy.Utilize gene, the contained information of genome to infer that the character of proteins encoded just becomes antigen protein and epi-position identification and the shortcut of identifying.The nucleotide sequence of three large nucleic acids sequence library GenBank, DDBJ in the world and EMBL accumulative total surpasses hundred million at present, and the base number surpasses 10,000,000,000 bp, and is still increasing rapidly.

Summary of the invention

The objective of the invention is to make full use of abundant nucleic acid and protein sequence resource, catch the proteic mimic epitopes of unknown pathogeny microbial antigen, i.e. the information biology screening method of pathogenic micro-organism mimic epitopes fast by specific bioinformatic analysis method.

The present invention,, is verified to differentiate the validity of bioinformatics method with immunological method by information biology screening mimic epitopes as model animals then with lymphocystis disease virus different isolates (LCDV-1, LCDV-cn).Basic technological line is: the genome sequence that utilizes the LCDV-1 (lymphocystis disease virus Europe strain isolated) that has measured; LCDV-cn (Chinese pathogenic strain with genome and antigen protein the unknown; be about 77% with the homology of LCDV-1) be object, by the bioinformatic analysis of LCDV-1 being predicted protective antigen encoding gene and the mimic epitopes thereof of LCDV-cn.

The principle of present method institute foundation mainly is the consistence of biological phenomena height, particularly all biological elementary cells (deoxyribonucleotide, ribonucleotide, amino acid) that constitute carrier of genetic information nucleic acid and physiological function carrier protein do not have difference on structure, composition, and do not have different on the composition of the functional unit genetic codon of genetic expression and encoding function yet.

Present method is utilized known nucleic acid and protein sequence, infer according to the distinctive feature structure of protein families territory only to contain portion gene or genome sequence column information and the nearly edge pathogenic micro-organism antigen protein mimic epitopes of its encoded protein matter biological function the unknown, and in addition immunology checking.Though present method is the information biology analytic target with the LCDV-1 by known group sequence only; infer the LCDV-cn protective antigen mimic epitopes of genome and the unknown of genes involved sequence; but be based on the consistence of above-mentioned nucleic acid, protein structure function, so the pervasive screening of the pathogenic micro-organism mimic epitopes of known or unknown gene, genome sequence of present method in other.

Description of drawings

Fig. 1 is that the competitive inhibition enzymoimmunoassay of mimic epitopes 29B is checked immunogenic data plot

Wherein, A-F: be respectively monoclonal antibody YpH4A1,3,4,7,9,10;

G-H: be respectively monoclonal antibody YpH4B1,5.

YpH4A1-10 is the LCDV-cn specific monoclonal antibody, and YpH4B1,5 is irrelevant monoclonal antibody.

Polypeptide (mimic epitopes) shown in the figure in the reaction is 29B (all the other is unlisted).

X-coordinate among the figure: BC: blank group; NCa: negative control group (add polypeptide, do not add corresponding monoclonal antibody); NCb: negative control group (replacing monoclonal antibody) with mouse IgG; PC group: positive control (only add corresponding monoclonal antibody, do not add polypeptide).100,50,10,1: corresponding to peptide concentration, unit is μ g/ml.

Ordinate zou among the figure: microplate reader is measured the optical density value of wavelength 450nm, and unit is OD.

Stylolitic part shown in the figure is the mean value and the standard deviation (shown in the strigula of post figure top) of experimental group.

Used monoclonal antibody, antibody concentration are 100 μ g/ml.

The ELISA response procedures: (LCDV-cn0.5 μ g bag is spent the night for 4 ℃ by 96 orifice plates antigen, wash plate 3 times, the same ordinary method of control group application of sample, experimental group are established 8 multiple holes, the polypeptide of each concentration is hatched 1hr for 37 ℃ with the monoclonal antibody equal-volume earlier, is added in 96 orifice plates 37 ℃ then and hatches 1hr.Add horseradish peroxidase sheep anti mouse ELIAS secondary antibody and TMB (tetramethyl benzidine), 2M H ₂SO ₄Stop, (BioRad, Microplate Reader Model550) measure 450nm OD value to the microwell plate calculating instrument.

Embodiment

Present method as model animals, has at first been carried out the BLAST comparison to the LCDV-1 genome sequence with lymphocystis disease virus different isolates (LCDV-1, LCDV-cn), and therefrom screening obtains two protective antigen albumen candidate gene TK and ORF29; Then respectively to the protein structure of two genetic expressions with character has been carried out hydrophobic and wetting ability, antigenicity and stride the membrane structure analysis, comprehensive said structure property analysis result, the corresponding mimic epitopes of LCDV-cn of the calculation the unknown by comprehensive antigenic index at last.

The mimic epitopes of above-mentioned acquisition is carried out synthetic, check the immunoreactivity of various mimic epitopess by competitiveness enzyme-linked immuning adsorpting analysis.

Concrete mode is as follows:

1, the BLAST retrieval analysis of antigen protein

Analyze (http://www.ncbi.nlm.nih.gov/blast by LCDV-1 being carried out BLAST; parameter is got default value) and protein function identify (AACompSim:http: //www.expasy.ch/tools/aacsim/, PROPSEARCH:http: //www.embl-heidelberg.de/prs.html) preliminary screening LCDV-cn (Chinese pathogenic strain) protective antigen encoding gene.Through the homology analysis of nucleotide sequence and the functional analysis of expressing protein, determine that with Iridoviridae camber conservative TK gene and LCDV-1 envelope protein encoding gene ORF29 be the candidate gene of antigenic determinant analysis and screening.

2, information biology derivation antigen protein mimic epitopes

The TK of LCDV-1 and the proteins encoded of ORF29 have been carried out bioinformatic analysis with identical method, and the presuming method of mimic epitopes comprises following two portions:

2.1 the regional structural analysis of antigen protein:

Have several different methods to carry out structural analysis of protein at present, present method has optionally adopted following 12 kinds of bioinformatic analysis methods.Utilize these methods that the analytical results of two kinds of antigen proteins is listed in table 1, fiducial interval is 95%.

Structure and properties analytical procedure and the result thereof of table 1 antigen protein TK, ORF29

Protein structure and character	Existing basic skills	Fiducial interval (TK)	Fiducial interval (ORF29)
				Hydrophobicity hydrophily antigenicity is striden diaphragm area	Fauchere Manavalan Janin Kyte/Doolittle Sweet/Eisenberg GES Von Heijine Hopp-Woods Parker Protrrusion index Welling Von Heijine	0.4127，0.4610 12.85，12.92 -0.1112，-0.0796 -0.2056，-0.0694 0.0133，0.0609 0.9203，1.149 1.058，1.220 -0.1381，-0.0514 0.9517，1.266 4.241，4.298 -0.0360，-0.0270 -0.5671，-0.5265	0.3539，0.4197 12.80，12.91 -0.1753，-0.1244 -0.2929，-0.0826 -0.0882，-0.0267 1.114，1.422 1.050，1.268 0.0276，0.1461 1.483，1.905 4.327，4.400 -0.0284，-0.0197 -0.5710，-0.4997

2.2 synthetic antigen analysis

Complicacy based on protein structure is considered, and the difference of basic skills data acquisition modes in the table 1, the present invention is integrated above-mentioned 12 kinds of different protein structures, the obtained data of property analysis method, and the present invention provides unified comprehensive antigenic index I.The calculation formula of comprehensive antigenic index I is as follows:

$I=\mathrm{\Σ}\left\{a_i\stackrel{\‾}{x_j}\stackrel{\‾}{x_m}\sqrt{\left(n_j\mathrm{\Σ}{(x_{\mathrm{im}}-x_m)}^2\right)/\left[(n_m-1)\mathrm{\Σ}{(x_{\mathrm{ij}}-\stackrel{\‾}{x_j})}^2\right]}\right\}$

Wherein:

The comprehensive antigenic index of I=, a=structural analysis of protein method coefficient, i=sequence number, n _j=regional amino acid no, n _mAmino acid sum in the=protein.I＜0, non-antigen zone; I＞0 o'clock, antigenicity is remarkable, and the I value is high more, and antigenicity is remarkable more.According to this, infer 3 mimic epitopess of LCDV-cn, i.e. TK3 in the table 2,29A and 29B.

For further immunology checking, from gene TK and ORF29, oppositely infer each non-mimic epitopes in contrast specially respectively, see Table TK2 and 29C in 2.

The information biology prediction of table 2 mimic epitopes and control sequence

Gene/ORF	The mimic epitopes sequence	The position	The antigenicity aggregative index	Remarks
					TK TK1 TK2 TK3 ORF29 29A 29B 29C	AIKLKADNRMNNNGTIRWC ENSARLNEMGSEYYSLV TNEERRLMGT TKNNMSRTSETDNN CKKNSDSTDC KISLKAYDSK	99-106 348-364 252-261 129-142 164-173 307-316	1.309 -1.988 4.5763 7.4475 8.0769 -1.606	Canonical sequence control sequence mimic epitopes mimic epitopes mimic epitopes control sequence

The immune analysis of 3 mimic epitopess and evaluation

Mimic epitopes and contrast and canonical sequence according to bioinformatic analysis obtains have synthesized 6 corresponding polypeptide, acetylize sealing NH ₂-end has carried out competitive inhibition ELISA experiment with the specific monoclonal antibody of anti-LCDV-cn, with the immunoreactivity of check mimic epitopes.Result's (as shown in Figure 1) shows that the 29B that the antigenicity index is the highest all produces significant retarding effect, P＜0.0001 with monoclonal antibody YpH4A1, YpH4A3, YpH4A7 and the YpH4A10 of examining.No tangible competitive effect between other mimic epitopes and contrast, canonical sequence and confession inspection monoclonal antibody.Promptly there is not competitive effect in control antibodies (mouse IgG and irrelevant monoclonal antibody YpH4B1, B5), does not see cross reaction yet, illustrates for the inspection monoclonal antibody to have the LCDV-cn specificity.The result shows, from negative findings, canonical sequence (as sealing its epi-position because of the secondary structure that self produces without antigenic processing and the process of offering) and control sequence are as not competing property of expection retarding effect, and its reason remains to be analysed in depth to confirm its true negative.The reason that positive findings does not appear in inspected two other mimic epitopes (TK3,29A) has two, and A, forecasting sequence are not mimic epitopess, and B, confession inspection monoclonal antibody do not cover this epi-position.According to interpretation of result, the latter's possibility is bigger, because there are 4 kinds all with 29B the intensive competitive inhibition to take place in 6 kinds of monoclonal antibodies that supply to examine, they have overlapping epi-position different response intensity explanations, therefore can not react with the linear epitope that other sequence has nothing to do.

Positive findings offer some clarification on utilize bioinformatic analysis LCDV-1 genome to infer effective anesthesia needle the epi-position that the specific monoclonal antibody of LCDV-cn is discerned of mimic epitopes; because multiple monoclonal antibody all can produce significant restraining effect mutually with 29B; can infer that this epi-position should be the linear antigenic determinant in the protective antigen.

Mimic epitopes bioinformatic analysis and Forecasting Methodology that the present invention adopts prove feasible through immunological test, it is not only applicable to known or genome sequence are carried out antigenicity analysis, also can be used for the unknown gene/genome antigenicity analysis of the nearly higher species of intermarginal conservative degree simultaneously.Present method can replace traditional protein separation is identified and immediate development is got up phage display peptide library even protein biochip technology commonly used in the present mimic epitopes screening to a certain extent, great amount of manpower and material resources be can save, antigen protein and antigenic determinant thereof obtained simultaneously fast, efficiently.

Claims (2)

1, the information biology screening method of pathogenic micro-organism mimic epitopes has at first carried out the BLAST comparison to the LCDV-1 genome sequence, and therefrom screening obtains two protective antigen albumen candidate gene TK and ORF29; Carried out hydrophobic to the protein of two genetic expressions respectively then and wetting ability, antigenicity and stride the membrane structure analysis, comprehensive 12 kinds of textural property analytical resultss, by the calculation formula (I) of comprehensive antigenic index, wherein, the sequence of I＞O is estimated as the LCDV-cn mimic epitopes of the unknown at last.

2, the information biology screening method of pathogenic micro-organism mimic epitopes is characterized in that above-mentioned comprehensive antigenic index I, and the calculation formula of I is as follows:

$I=\mathrm{\Σ}\{a_i\stackrel{\‾}{x_j}/\stackrel{\‾}{x_m}\sqrt{\left(n_j\mathrm{\Σ}{(x_{\mathrm{im}}-x_m)}^2\right)/\left[(n_m-1)\mathrm{\Σ}{(x_{\mathrm{ij}}-\stackrel{\‾}{x_j})}^2\right]}\}$

Wherein:

The comprehensive antigenic index of I=, a=structural analysis of protein method coefficient, i=sequence number, n _j=regional amino acid no, n _mAmino acid sum in the=protein.

Images