抗原多肽的設(shè)計、偶聯(lián)策略

1、.：.；抗原多肽的設(shè)計、偶聯(lián)戰(zhàn)略抗體是生命科學(xué)研討中不可或缺的工具之一，運用范圍包括蛋白質(zhì)表達檢測和鑒定、蛋白質(zhì)加工、蛋白質(zhì)在細胞內(nèi)的定位、免疫中和反響、蛋白質(zhì)同源構(gòu)造域研討、蛋白質(zhì)純化以及疾病的免疫診斷和治療。雖然抗體的制備過程不存在技術(shù)難點，但是抗原的選擇以及所制備抗體的用途對能否獲得一個優(yōu)質(zhì)高效的抗體至關(guān)重要。以下將對抗原多肽的設(shè)計、偶聯(lián)戰(zhàn)略等逐一引見.抗原設(shè)計 首先選擇適宜的多肽序列，明確最終產(chǎn)物的用途對選擇序列非常重要。假設(shè)僅僅需求消費針對蛋白質(zhì)某個區(qū)域的特異抗體，比如研討蛋白質(zhì)N端的前提物，我們就需求設(shè)計N末端的多肽抗原。假設(shè)抗體的運用目的是識別修飾的氨基酸，如磷酸化的絲氨酸、蘇氨

2、酸或者酪氨酸，乙?；嚢彼岬龋捅匦鑼Χ嚯倪M展相應(yīng)的修飾。假設(shè)抗體最終用來識別自然形狀下的蛋白質(zhì)，對抗原的設(shè)計就要求更高。普通情況下抗血清可以識別用來免疫的多肽序列，但是不一定識別蛋白質(zhì)的折疊構(gòu)造。蛋白質(zhì)的抗原決議簇普通由612個氨基酸構(gòu)成，呈延續(xù)性或者非延續(xù)性序列。延續(xù)性抗原決議簇由延續(xù)的氨基酸序列構(gòu)成，而非延續(xù)抗原決議簇包括一組非延續(xù)氨基酸，這些氨基酸由于蛋白質(zhì)的折疊而構(gòu)成在空間上相互毗鄰。針對延續(xù)性抗原決議簇的抗體可以識別沒有被埋藏在蛋白質(zhì)內(nèi)部的序列，而非延續(xù)性抗原的抗體能否識別抗原決議簇取決于用于抗體消費的多肽能否存在二級構(gòu)造。氨基酸序列的親水性、流露性、柔韌性決議了多肽的抗原性。許多

3、水融性的自然形狀下的蛋白質(zhì)其親水序列暴露在外測，而疏水性氨基酸序列包埋在內(nèi)部?？贵w結(jié)合蛋白質(zhì)外表的抗原決議簇，另外抗原決議簇柔韌性比較高。蛋白質(zhì)的C末端經(jīng)常暴露在外測并且有較高的柔韌性，因此經(jīng)常被用來作為抗體消費的抗原。但是假設(shè)C末端是跨膜蛋白質(zhì)的膜內(nèi)部分，該序列能夠由于疏水性太強而不適宜用來作為抗原。同C末端序列類似，蛋白質(zhì)的N末端序列也經(jīng)常暴露在蛋白質(zhì)的外表，同樣為首選抗原序列。預(yù)測蛋白特性例如親水性、疏水性及二級構(gòu)造例如螺旋，折疊，盤旋的一些算法有助于選擇流露性較高，有抗原性的內(nèi)部序列以用于抗體生成。常見預(yù)測性算法有如下三種，Hopp及Woods所描畫的親水性曲線給蛋白序列中的每一個氨基

4、分配一個平均親水性值，對于一系列的相鄰氨基，平均親水性的最高點通常就位于抗原決議簇或在其附近。Kyte 及 Doolittle 所提出的另一算法略有不同，它主要是衡量蛋白序列的親水性及疏水性趨勢，該算法對于預(yù)測某蛋白的外部及內(nèi)部區(qū)域非常有用。蛋白的二級構(gòu)造那么可以經(jīng)過CHOU/FASMAN 或 LIM 所提出的算法來預(yù)測。流露性或易接近區(qū)經(jīng)常和螺旋區(qū)或延展的二級構(gòu)造區(qū)相鄰。并且，具有盤旋或雙性螺旋特性的序列區(qū)也具有較好的抗原特性。目前有許多商用軟件包都運用了這些不同的算法，例如MacVectorTM，DNAStarTM及PC-GeneTM。要想預(yù)測準確，不能只運用一種算法。結(jié)合各種不同的預(yù)測方

5、法來預(yù)測抗原性區(qū)域，可使勝利率大大提高?？乖詤^(qū)域確定以后，接下來要確定多肽的長度。關(guān)于選定多肽長度有兩種不同的觀念。一種觀念以為長的多肽2040個氨基酸比較好，由于長的多肽無疑會添加抗原基的數(shù)量。另一種觀念那么以為小的多肽更有效，運用小的多肽能保證所產(chǎn)生抗體的位點特異性。但有一點是明確的，不論長度如何，所選多肽必需可以較容易地經(jīng)過生化合成得到，并且能溶解到水溶性緩沖劑進展載體蛋白的耦聯(lián)。由于受副反響的影響較大，高于二十個氨基酸的多肽通常很難進展高純度合成，并且經(jīng)常會含有缺失性序列。另一方面，太短的多肽10kd進展?jié)饪s，以備測試；利用0.2M氨基乙酸glycine進展洗脫pH12，在pH2.0

6、時開場洗脫，在吸光度降到基線以下時搜集洗脫液。將洗脫液的pH降低0.5-1，直到已沒有可檢測到的抗體從柱上洗下來；搜集從純化柱上搜集的各個部分，搜集后經(jīng)過參與1/10體積1 M Tris-HClpH=8.0 或pH 8.0 的洗脫液將其中和；將純化的多肽進展ELISA分析，以決議具有最高親和性的洗液部分。將適當?shù)目贵w洗脫液濃縮至2-5mg/ml，混合50甘油在20oC下保管，或混合0.1% BSA在4oC下保管。參與0.01% NaN3作為防腐劑。3看起來有些渾濁的血清能否還可以運用？普通來講，這不會是問題。運用前可以用0.2m的過濾器去除血清中的小顆粒Antigen design & ser

7、a purificationAntibodies to small peptides have become an essential tool in life science research, with applications including gene product detection and identification, protein processing studies, diagnostic tests, protein localization, active site determination, protein homology studies and protei

8、n purification. While it is quite easy to generate anti-peptide antibodies, it is important to carefully consider the ultimate use for the antibody and the sequence used to ensure success. This tech sheet will briefly explore peptide selection and design, coupling strategy, and carrier proteins whic

9、h are important factors in anti-peptide antisera generation. Serum purification will also be discussed. For more complete coverage of antigen design, please refer to the References1,2. Peptide Selection and DesignThe first step in the process is the selection of the appropriate peptide sequence. At

10、this step the ultimate use for the antibody must be considered. If the antibody is needed to probe a specific protein domain then the choice is simple. For example, if one is studying proteolytic processing of an N-terminal precursor, antibodies against the N-terminal region of interest would be rai

11、sed. Likewise if the goal is to monitor the phosphorylation state of a specific sequence, antibodies to the phosphorylated sequence can be used. If the goal is to raise antibodies that will recognize the protein in its native state, the problem becomes more complex. Anti-peptide antibodies will alwa

12、ys recognize the peptide. However, the same antibody may not recognize the sequence within the folded intact protein. Sequence epitopes in proteins generally consist of 6-12 amino acids and can be classified as continuous and discontinuous. Continuous epitopes are composed of a contiguous sequence o

13、f amino acids in a protein. Anti-peptide antibodies will bind to these types of epitopes in the native protein provided the sequence is not buried in the interior of the protein. Discontinuous epitopes consist of a group of amino acids that are not contiguous but are brought together by folding of t

14、he peptide chain or by the juxtaposition of two separate polypeptide chains. Anti-peptide antibodies may or may not recognize this class of epitope depending on whether the peptide used for antisera generation has secondary structure similar to the epitope and/or if the protein epitope has enough co

15、ntinuous sequence for the antibody to bind with a lower affinity. When examining a protein sequence for potential antigenic epitopes, it is important to choose sequences which are hydrophilic, surface-oriented, and flexible3. Most naturally occurring proteins in aqueous solutions have their hydrophi

16、lic residues on the surface and their hydrophobic residues buried in the interior. Antibodies bind to epitopes on the surface of proteins. Additionally, it has been shown that epitopes have a high degree of mobility4. Because the C-termini of proteins are often exposed and have a high degree of flex

17、ibility they are usually a good choice for generating anti-peptide antibodies directed against the intact protein. If the protein is an integral membrane protein and the C-terminus is part of the transmembrane segment, this sequence will be too hydrophobic and not a good choice. Like the C-terminus,

18、 the N-terminus is also frequently exposed and on the surface of the protein making it an ideal candidate for antibody generation. If a protein sequence is derived from the cDNA sequence, the leader sequence should not be included in the sequence selected for antibody generation. Algorithms for pred

19、icting protein characteristics such as hydrophilicity/hydrophobicity and secondary structure regions such as alpha-helix, beta-sheet and beta-turn aid selection of a potentially exposed, immunogenic internal sequence for antibody generation. Hydrophilicity plots as described by Hopp and Woods5 assig

20、n an average hydrophilicity value for each residue in the sequence. The highest point of average hydrophilicity for a series of contiguous residues is usually at or near an antigenic determinant. A slightly different algorithm described by Kyte and Doolittle6 evaluates the hydrophilic and hydrophobi

21、c tendencies of the sequence. This profile is useful for predicting exterior vs. interior regions of the native protein. Secondary structure can be identified by the use of algorithms developed by Chou and Fasman7 or Lim8. Surface regions or regions of high accessibility often border helical or exte

22、nded secondary structure regions. In addition, sequence regions with beta-turn or amphipthic helix character have been found to be antigenic9. Many commercial software packages such as MacVectorTM, DNAStarTM, and PC-GeneTM incorporate these algorithms. To be sucessful, none of the algorithms should

23、be used alone. Combined use of the predictive methods may result in a success rate as high as 86% in predicting antigenic determinants9,10. Once the protein region of interest has been identified, the length of the peptide must be selected. There are two differing thoughts on the topic of peptide le

24、ngth. One suggests that long peptides (20-40 amino acids in length) are optimal because it increases the number of possible epitopes. The other suggests that smaller peptides are sufficient, and their use ensures that the site-specific character of anti-peptide antibodies is retained. Clearly, any p

25、eptide selected must be chemically synthesizable and should be soluble in aqueous buffer for conjugation to the carrier protein. Peptides longer than 20 residues in length are often more difficult to synthesize with high purity because there is greater potential for side reactions, and they are like

26、ly to contain deletion sequences. On the other hand, short peptides (10 amino acids) may generate antibodies that are so specific in their recognition that they cannot recognize the native protein or do so with low affinity. The typical length for generating anti-peptide antibodies is in the range o

27、f 10-20 residues. Peptide sequences of this length minimize synthesis problems, are reasonably soluble in aqueous solution and may have some degree of secondary structure. Coupling StrategyA factor that is often over-looked when designing a synthetic peptide is the method of coupling the peptide to

28、the carrier protein. For example, N-terminal sequences should be coupled through the C-terminal amino acid and vice versa for C-terminal sequences. Internal sequences can be coupled at either end. Another consideration for internal sequences is to acetlyate or amidate the unconjugated end as the seq

29、uence in the native protein molecule would not contain a charged terminus. The most common coupling methods rely on the presence of free amino (alph-amino or Lys), sufhydryl (Cys), or carboxylic acid groups (Asp, Glu, or alpha-carboxyl). Coupling methods should be used that link the peptide to the c

30、arrier protein via the carboxy- or amino-terminal residue. The sequence chosen should not have multiple residues that may react with the chosen chemistry. If multiple reactive sites are present, try to shorten the peptide or choose the sequence so they are all localized at either the amino or the ca

31、rboxyl terminus of the peptide. For internal sequences the end furthest from the predicted epitope is normally favored as this avoids potential masking problems. The EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) or carbodiimide method is routinely used in the Sigma-Genosys labor

32、atory unless otherwise stated by the researcher. Carbodiimides can activate the side chain carboxylic groups of aspartic and glutamic acid as well as the carbooxyl terminal group to make them reactive sites for coupling with primary amines. The activated peptides are mixed with the carrier protein t

33、o produce the final conjugate. If the carrier protein is activated first, the EDC method will couple the carrier protein through the N-terminal alpha amine and possibly through the amine in the side-chain of Lysine, if present in the sequence. The m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)

34、is a heterobifunctional reagent that can be used to link peptides to carrier proteins via cysteines. The coupling takes place with the thiol group of cysteine residues. If the chosen sequence does not contain Cys it is common to place a Cys residue at the N- or C-terminus to obtain highly controlled

35、 linking of the peptide to the carrier protein. For synthesis purposes we recommend that the placement of cysteine be at the N-terminus of the peptide if possible. Glutaraldehyde is a bifunctional coupling reagent that links two compounds through their amino groups. Glutaraldehyde provides a highly

36、flexible spacer between the peptide and carrier protein for favorable presentation to the immune system. Unfortunately, glutaraldehyde is a very reactive compound and will react with Cys, Tyr and His to a limited extent. The result is a poorly defined conjugate. The glutaraldehyde method is particul

37、arly useful when a peptide contains only a single free amino group at its amino terminus. If the peptide contains more than one free amino gorup, large multimeric complexes can be formed, which are not well defined, but are highly immunogenic. Selecting the Protein CarrierConjugation to a carrier pr

38、otein is important because peptides are small molecules, that alone do not tend to be immunogenic, thus possibly eliciting a weak immune response. The carrier protein contains many epitopes that stimulate T-helper cells, which help induce the B-cell response. Many different carrier proteins can be u

39、sed for coupling to synthetic peptides. The most commonly selected carriers are keyhole limpet hemacyanin (KLH) and bovine serum albumin (BSA). The higher immunogenicity of KLH often makes it the preferred choice. Another advantage of choosing KLH over BSA is that BSA is used as a blocking agent in

40、many experimental assays. Because antisera raised against peptides conjugated to BSA will also contain antibodies to BSA, false positives may result. Although KLH is large and immunogenic, it may precipitate during cross-linking, making it difficult to handle in some cases. Ovalbumin (OVA) is anothe

41、r useful carrier protein. It is a good choice as a second carrier protein when verifying whether antibodies are specific for the peptide alone and not the carrier. Rabbit Serum Albumin (RSA) may be used when the antibody response to the carrier protein must be kept to a minimum. Rabbits immunized wi

42、th RSA conjugate are less likely to raise antibodies to the carrier, as the RSA is recognized as self. If the RSA conjugate were injected into another host, the protein would not be recognized as self. It is important to recognize that the immune system reacts to the peptide-protein carrier as a who

43、le and that there will be a portion of response directed against the conjugated peptide as well as the linker and the carrier protein1. When screening by ELISA it is advisable to use a peptide conjugate prepared using a different carrier protein. This is not necessary if performing ELISA assays wher

44、e the plates are coated directly with unconjugated peptide. Multiple Antigenic Peptides (MAPs)The MAP system represents a unique approach to anti-peptide antibody generation11. The system is based on a small immunogenically inert branched lysine core onto which multipe peptides are synthesized in pa

45、rallel. The result after synthesis is a three-dimensional molecule, which has a high molar ratio of peptide antigen to core molecule and therefore does not require the use of a carrier protein to induce an antibody response. Each core molecule may contain four identical peptides. In theory, MAP has

46、an advantage when compared to its monomeric counterpart attached to a carrier protein in that the lysine core of a MAP is small compared with the peptide antigen. Therefore, the concentration of antigen is at a maximum. The result is a highly immunogenic MAP, which exhibits significantly higher tite

47、rs when compared to its monomeric counterpart attached to a carrier protein. It should be noted that there are some synthesis concerns when making a MAP complex. The branched nature of the lysine core allows for multiple copies of the peptide to be synthesized; however, steric hindrance becomes a pr

48、oblem during the synthesis of long peptides, resulting in some arms of the dendrimer being deletion products. The high molecular weight of the complex does not lend itself to good quality control measures (mass spec and/or analytical HPLC). An indirect synthesis of the MAP can eliminate analysis pro

49、blems. In the indirect method, the peptide is first synthesized, purified then analyzed using mass spec and analytical HPLC. The peptide antigen is then coupled through a Cys to a functionalized lysine core. Choice of HostWhen attempting to raise an antibody, choose an animal that is genetically ver

50、y different from the source of immunogen. In order to achieve maximum immune response, it is important to avoid self-recognition of the immunogen by the host animal. As an example, when raising antibodies against a human protein, it is more suitable to use a rabbit or mouse host than a monkey. For h

51、ighly conserved mammalian proteins, raising antibodies in the avian (chicken) system is often a preferred alternative. Adjuvant, Immunization, & Sera CollectionSigma-Genosys routinely uses Freunds adjuvant for immunization purposes. The first injection is given in Complete Freunds adjuvant. Adjuvant

52、 is combined with the antigen to improve the immune response so that less vaccine is needed to produce more antibodies. The adjuvant allows a slow release of the antigen which allows for continual stimulation. Injections are routinely performed subcutaneously at multiple sites. A pre-immune bleed sh

53、ould be drawn from each host animal to produce a baseline to which the production bleeds can be compared. The drawn sera will contain a number of different types (IgG, IgM, IgA) and subclasses (Ig1, Ig2a, Ig2b, Ig3). Sodium azide (0.1%) can be added to the sera. Sodium azide is a broad-spectrum enzy

54、me inhibitor and acts as an antimicrobial agent. Sodium azide should not be added to sera when using in cell culture or in vivo studies. Antisera PurificationIf a high background is observed in assays using the antisera, various purification techniques are available. It is important to first check t

55、hat the background is non-specific and not due to the response against the peptide. This can be determined by performing a competitive peptide blocking study. Peptide blocking studies check that the response against the target protein is not a background artifact. Ammonium Sulfate PrecipitationAmmon

56、ium sulfate precipitation is a commonly used method for removing protein from solution. The method is a fairly crude, non-specific purification that removes the majority of plasma proteins and leaves the immunoglobulin fraction. When in solution, proteins form hydrogen bonds with water through their

57、 exposed polar and ionic groups. Adding small ions such as ammonium or sulfate removes water molecules from the protein, resulting in precipitation of the protein out of solution. It should be stated that ammonium sulfate precipitation will not result in highly purified antibodies. The contaminants

58、will consist of other high-molecular-weight proteins and proteins that are trapped in the large flocculent precipitates. It is recommended that ammonium sulfate precipitation be used as part of a purification scheme involving further purification steps. Protein A/GProtein A or Protein G purification

59、 removes the IgG fraction based on the specificity of these proteins for the Fc portion of the IgG. Protein A is produced from Staphylococcus aureus. It has the capacity to bind at least two molecules of IgG. The binding is specific to the Fc portion and does not affect the antigen binding sites. Protein G is isolated from Group G streptococci and binds the Fc region of the IgG in a similar manner to Protein A. Protein A a