HBV G145R突变对S蛋白及“a”决定簇合成肽抗原性影响

人工合成G145R突变后HBsAg"a"决定簇多肽G145R-SP24,将G145R-SP24、重组G145R变异S蛋白及重组野生型HBsAg分组免疫BALB/c小鼠,制备相应抗血清。通过观察各免疫原与抗血清或抗G145R变异HBsAg McAb的免疫反应特征,分析并比较G145R突变后对S蛋白及"a"决定簇合成肽抗原性的影响。结果,几种免疫原均取得理想的免疫效果,ELISA证实各抗血清均有较好的特异性。G145R-SP24和G145R变异S蛋白免疫小鼠获得的抗血清效价较野生型HBsAg略低,且抗原性也发生了明显改变,G145R突变后"a"决定簇仍然具有较好的免疫原性;另外G145R-SP24与G145R变异S蛋白之间也显示出不同的抗原性,提示孤立的G145R变异HBsAg"a"决定簇与完整G145R S蛋白的"a"决定簇可能形成不同的空间构象。本研究为进一步探讨"a"决定簇突变后空间结构的变化规律与抗原性改变的特点奠定一定的基础。     

G145R变异乙型肝炎表面抗原诱导的抗体性质研究

乙型肝炎表面抗原（HBsAg）变异可导致抗原性改变。HBsAga决定簇最常见的变异位点是G145R。G145R变异可能改变原来的抗原表位，并形成新的抗原表位。本研究探讨G145RHBsAg能否刺激机体产生抗体及免疫应答的情况。     

乙型肝炎病毒表面抗原基因点突变对HBsAg抗原性的影响

目的 研究乙型肝炎病毒表面抗原(HBsAg)常见基因点突变对HBsAg抗原性的影响,了解我国目前常用的HBsAg检测试剂对HBV S基因突变株的检测灵敏性,减少漏检,有效控制乙肝病毒(HBV)感染的传播.方法 构建HBsAg重组野毒株和重组变异株表达质粒,分别将重组野毒株HBsAg表达质粒pSS1adw2及pSS1adr和重组变异株HBsAg表达质粒pSS1adw2-145Arg、pSS1adr-126 Ser和pSS1adr-126 Asn转染COS-7细胞,进行瞬时转染.采用市售HBsAg ELISA检测试剂盒对细胞上清进行抗原性检测.结果 野毒株HBsAg和两种126位变异株HBsAg具有较好的抗原性;145位点突变后、导致HBsAg的抗原性下降.结论 推测是由于145位点变异影响了"a"抗原决定簇的空间结构,从而降低了其与抗-HBs的结合能力.     

大鼠P450侧链裂解酶抗原决定簇多参数预测及合成

目的预测大鼠P450侧链裂解酶（P450scc）的抗原表位,确定合成肽序列,并合成八分枝肽,制备多克隆抗体。方法根据P450scc的氨基酸序列,采用GOLDKEY核酸及蛋白质分析软件,多参数预测P450scc的抗原表位,筛选并确定合成肽序列。采用的方案包括抗原性、Hopp＆Woods亲水性、可及性、柔韧性、电荷分布参数、二级结构β-转角和万氏综合表位参数方案。利用固相合成法,在Rink树脂上合成八分枝赖氨酸核心树脂,并在此基础上合成八分枝肽,用于制备抗体。结果将P450scc的羧基端16肽确定为合成肽序列（LKQDLGSTMPRKGDTV）,约Mr8000。固相合成获得目的肽约30mg,经鉴定纯度达到90％以上。结论P450scc抗原表位预测及八分枝肽固相合成,为利用该合成肽制备抗体提供了一条简捷的途径。     

利用噬菌体肽库筛选IL-2Rα模拟表位肽的研究

目的 筛选IL-2Rα模拟表位肽,为研制高效、特异性强的小分子肽类免疫抑制剂奠定基础.方法 应用离子交换层析法从小鼠腹水中纯化抗人IL-2Rα单克隆抗体5G1,细胞ELISA方法检测其特异性.用5G1单抗筛选噬菌体环七肽库,并对噬菌体克隆进行抗原性鉴定.根据阳性克隆的DNA序列合成环肽并鉴定其生物活性.结果 经5轮筛选、鉴定,获得5个与5G1有较强结合特性的噬菌体阳性克隆.DNA测序并推导氨基酸序列,得到Lys-X-{X}-Lys-Gly保守序列.依此序列合成环七肽CP,小鼠淋巴细胞增殖试验证实其有免疫抑制活性.结论 环肽CP为IL-2Rα模拟表位肽,可作为IL-2Rα的拮抗剂发挥免疫抑制效应.     

小鼠抗人C5aR短肽（9—30）单克隆抗体制备及鉴定

目的获得有生物功能的小鼠抗人 C5 a R短肽单克隆抗体。方法对 C5 a受体 (CD88)二级结构和 B细胞表位进行分析研究 ,采用 Fmoc方案固相合成 C5 a R N-端第 9～ 30位氨基酸残基的 2 2 -肽 ,以此为抗原免疫 Balb/ c小鼠。结果建立了 1株小鼠抗人 C5 a R短肽杂交瘤细胞系 E3,其平均染色体数目为 10 2条 ,所分泌抗体为 Ig G1,κ型 ,腹水抗体效价为 1×10 - 4 ～ 1× 10 - 6 ,可识别 U 937、人脐静脉内皮细胞 (VEC)和 PMN等表达 C5 a R细胞。 E3单抗的亲和常数 Ka=2 .5× 10 5 ,其结合表位为 C5 a R第 15～ 2 1位氨基酸基序 D1 5 DKDTL2 0 D。结论 B细胞表位多肽具有免疫原性 ,可制作单克隆抗体 ,为 C5 a-C5 a R相互作用的研究以及 AL I、ARDS等 C5 a相关疾病的研究提供实验材料。     

小鼠抗人细胞色素P450 1A2合成肽抗血清的制备与鉴定

目的：制备小鼠抗人细胞色素P450 1A2（CYP1A2）抗体及其特性鉴定。方法：利用生物信息学方法分析CYP1A2蛋白的序列,根据4个指标（亲水性、柔韧性、抗原性及表面性）选择欲合成的多肽序列,设计、合成CYP1A2多肽。将其与载体蛋白钥孔戚血蓝素（keyhole limpet hemocyanin,KLH）偶联,免疫BALB/c雌性小鼠制备抗CYP1A2抗体。用间接ELISA法测定抗体的效价,Western blot鉴定其特异性。结果：成功地获得针对小分子CYP1A2的抗体。该抗体效价为1∶16000,可与CYP1A2合成肽及天然CYP1A2出现特异性反应。Western blot的结果显示,该抗体识别的相应抗原的相对分子质量（Mr）为58000。结论：合成的多肽-KLH复合物具有免疫原性,可用于制备相应的抗体。制备的小鼠抗CYP1A2合成肽抗体的效价高及特异性良好。     

LPS表位模拟肽诱导小鼠保护性免疫的研究

目的 鉴定三种LPS模拟位合成肽的抗原性、诱导抗LPS抗体的产生及对细菌攻击的保护性.方法 三种LPS模拟位合成肽与载体BSA交联后,ELISA测定其与多种抗LPS单、多克隆抗体的结合活性;合成肽与蓝载体交联物免疫Balb/c小鼠,观察其诱发小鼠体内抗LPS抗体产生的规律及血清抗LPS抗体效价;鉴定针对细菌感染的保护性效应. 结果 三种合成肽均能与抗鼠伤寒和大肠杆菌LPS抗体结合;用合成肽-蓝载体交联物免疫动物可诱发小鼠体内针对两种LPS的抗体反应,并对细菌攻击的免疫动物具不同程度的保护性作用,以合成肽13a,13b及12W与蓝载体交联物免疫的小鼠在鼠伤寒沙门氏菌攻击后存活时间分别为(6.5±0.77),(9.5±1.38)及(9.3±0.75) d,而PBS/蓝载体对照组存活时间为5 d.结论 本研究所采用的3种LPS表位模拟肽作为疫苗免疫小鼠能够产生针对细菌攻击的保护性效应,提示此3种表位模拟肽作为LPS交叉保护性疫苗候选的可行性与可靠性.     

常见乙型肝炎病毒表面抗原突变体的抗原性分析

目的：研究乙型肝炎病毒（HBV）表面抗原突变株和野毒株对表面抗体结合能力的差异,探讨免疫漏检基因变异株的机制及对策。方法：应用基因重组技术构建HBsAg常见基因突变株和野毒株表达质粒,分别在COS－7细胞中瞬时表达,定量后用常规HBsAg免疫诊断试剂盒检测,观察重组抗原与不同抗－HBs的结合能力。结果：T126 S变异与单克隆抗体的结合力增高,G145R,K141 E和2株三位点同时变异株则明显下降,对M133T变异与单克隆抗体的结合力影响不大。变异株与多克隆抗体的结合力也有变化,但仍能检测到大部分变异株抗原。结论:“a”抗原决定簇氨基酸的置换影响了HBsAg与抗－HBs的结合能力,并且氨基酸的置换位置和特性对抗原性的影响各有不同。因此,建议应用HBsAg多克隆抗体或开发研制“免疫逃逸突变株”的特异性抗体,以完善HBsAg免疫诊断试剂的抗体组成,减少免疫诊断对常见基因变异株的漏检。     

抗HCV HVR1模拟合成肽单克隆抗体的制备及特性鉴定

目的:制备抗-HCV高变区1(HVR1)合成肽单克隆抗体(mAb),并对其特性进行鉴定。方法:以固相合成的HCV HVR1合成肽与牛血清白蛋白(BSA)偶联后,免疫8周龄的BALB/c小鼠。采用杂交瘤技术,取免疫小鼠的脾细胞与骨髓瘤细胞融合,制备抗-HCV HVR1 mAb。经HAT、HT选择培养及有限稀释法进行克隆化后,用相关的试剂盒鉴定小鼠mAb的Ig亚类;经中和抑制后用ELISA法检测mAb的特异性;用间接ELISA法检测mAb腹水的效价及相对亲和常数。结果:获得1株可分泌抗-HCV HVR1合成肽特异性mAb的杂交瘤细胞1A9G9F11。该株mAb的Ig亚类为IgG3;腹水效价为3.125×10-5;相对亲和常数为1.0×106L/mol。该株mAb与HBsAg、HBeAg、HBcAg、HAAg、牛血清白蛋白、酪蛋白和胸腺5肽均无交叉反应,与HCV HVR1合成肽-牛血清白蛋白出现特异性反应。结论:成功地建立了1株可分泌抗-HCV HVR1 mAb的杂交瘤细胞1A9G9F11,为进一步的实验研究提供了重要的制剂。     

Mutant hepatitis B virus surface antigens (HBsAg) are immunogenic but may have a changed specificity

Mutant hepatitis B virus with substitutions within the coding region for HBV surface antigen (HBsAg) has been found naturally in chronic carriers. It is therefore important to clarify whether the identified substitutions within the HBsAg have impact on the antigenicity and immunogenicity of HBsAg. A total of nine mutated HBV s-genes with single representative mutations were generated by site-directed mutagenesis and subcloned into an expression vector. The binding of polyclonal and monoclonal antibodies to these mutant HBsAg (mtHBsAg) was tested by immunofluorescence (IF) staining of cells transfected with the expression vectors. The amino acid (aa) substitutions like G145R, F134S, and C147W affected the binding of anti-HBs antibodies to corresponding mtHBsAg to different extents. The impact of aa substitutions G145R and F134S on the immunogenicity was accessed by genetic immunization of mice with vectors expressing middle HBsAg with the corresponding mutations. The immunized mice developed antibodies to recombinant HBsAg containing the HBV preS region and HBsAg-specific cytotoxic T-cell. However, the development of antibody response to wild-type small HBsAg was significantly impaired by the aa substitutions in HBsAg. Based on this fact, we further investigated whether the mtHBsAg with the aa substitution G145R is able to induce mutant-specific antibody responses. Strikingly, serum samples from mice immunized with mtHBsAg with G145R recognized plasma-derived mtHBsAg. Two mouse MAbs specific to mtHBsAg were generated. One MAb recognized mtHBsAg with G145R but not wild type and other mtHBsAg. We conclude that HBsAg with aa substitutions are immunogenic but may have a changed fine specificity.     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.     

Immunological studies of the disulfide bridge region of Pseudomonas aeruginosa PAK and PAO pilins, using anti-PAK pilus and antipeptide antibodies.

Pseudomonas aeruginosa is an opportunistic pathogen that attaches to host cells via their pili. The pilus of P. aeruginosa PAK consists of a polymer of a single subunit, pilin, which is a 144-residue polypeptide. The C-terminal end of this protein is semiconserved in a number of strains and contains a disulfide bridge. We have synthesized the C-terminal peptide PAK (128-144)-OH in both its reduced and oxidized forms and the analog PAK(A-129) (128-144)-OH, in which cysteine-129 was substituted by alanine. These three peptides were used to immunize rabbits and prepare antipeptide antisera. It was found that antipeptide antisera to reduced peptide (17-R) and to oxidized peptide (17-O) bound to native PAK pili and cross-reacted with strain PAO pili in direct enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. However, the antiserum to the peptide immunogen PAK(A-129)(128-144)-OH, which does not have the ability to form the disulfide bridge, did not bind to either PAK or PAO pili. Competitive ELISA experiments with reduced and oxidized peptides of Ac-PAK(128-144)-OH showed that there was no difference in binding between the two peptides for 17-R or 17-O immunoglobulin G. When immunoglobulin G from native PAK antipilus antiserum was used in competitive or direct ELISA experiments, there was also no preference in binding to reduced or oxidized Ac-PAK(128-144)-OH or to PAK(A-129)(128-144)-OH. This result showed that the disulfide bridge in Pseudomonas pili is not critical to the immunogenicity of this region. However, the disulfide bridge is important in the immunogenicity of the C-terminal peptide when preparing antipeptide antisera that are cross-reactive with pili from different strains, since only the disulfide bridge peptide antisera cross-reacted well with the PAO pili as shown by competitive ELISA, suggesting that this region could be an important candidate for development of a synthetic vaccine.     

Transmission of G145R mutant of HBV to an unrelated contact

Household contacts of HBV-related chronic liver disease patients constitute a high-risk group for acquisition of HBV infection. Some of the HBsAg mutants are associated with liver disease and some are reported to be transmitted vertically. There is limited information on the horizontal transmission of Gly 145 Arg (G145R) mutant to related contacts. Its possible transmission to an unrelated third degree contact is reported in the present study. An HBV related chronic liver disease patient; the index patient, and his 11 household contacts were studied. This included four 1 degrees, three 2 degrees, one 3 degrees, and a sexual contact. Surface gene sequencing including the "a" determinant region was carried out in HBV DNA+ve subjects. The sequences were aligned and compared for the homology. HBV DNA was found to be positive in one 1 degrees, three 2 degrees, and one 3 degrees contact, besides the index patient. Histopathological studies revealed evidence of chronic hepatitis in all these contacts. Mutation T118V was present in all the six subjects. Mutant G145R along with T118V and T143M was identified in three subjects who included one 1 degrees, one 2 degrees, and one 3 degrees contact. Presence of T118V and T143M mutations along with G145R mutation in these subjects provides an indirect evidence for the possible horizontal transmission of G145R HBV variant to a 3 degrees unrelated contact. Of these three contacts with G145R mutation, only one 1 degrees contact was found to be HBsAg-ve. The data also reaffirms the earlier finding of HBsAg positivity in presence of G145R mutation of the S-gene. HBV exists as quasi-species and mixed population in subjects with chronic HBV infection.     

Mutated epitopes of hepatitis B surface antigen fused to the core antigen of the virus induce antibodies that react with the native surface antigen

Fusion of peptide epitopes to the core antigen (HBcAg) of hepatitis B virus (HBV) enhances their immunogenicity, both quantitatively and qualitatively. In a number of vaccine-induced mutants of HBV, glycine₁₄₅ of the surface antigen S polypeptide (HBsAg) has been replaced by arginine, resulting in loss of cross-reactivity with antibodies to normal (wild-type) HBsAg. HBcAg fusion proteins carrying the immunodominant epitope of HBsAg, in which glycine₁₄₅ was replaced by arginine, glutamic acid, or lysine, were produced in Escherichia coli and formed particles that displayed HBc antigenicity and immunogenicity similar to that of HBcAg itself. The fusion proteins also elicited T-cell-proliferative responsiveness to HBcAg and HBsAg. Fusions carrying either wild-type or mutated epitopes of HBsAg showed HBs antigenicity in immunoblot analysis and antigen-capture immunoradiometric assay, but both mutant and wild-type derivatives induced antibodies that cross-reacted with wild-type HBsAg. The results emphasise the potential for HBcAg fusion proteins in vaccines by broadening the antibody response in a way that could confer protection against both wild-type and variant forms of HBV. J. Med. Virol. 51:159–166, 1997. © 1997 Wiley-Liss, Inc.     

Mapping of linear B-cell epitopes of the S2 subunit of pertussis toxin.

The linear immunogenic and antigenic structure of the S2 subunit of pertussis toxin was investigated with synthetic peptides corresponding to regions of the protein sequence predicted to contain surface-exposed hydrophilic beta turns. Five peptides as peptide-bovine serum albumin conjugates were recognized by anti-pertussis toxin antiserum and were thus designated "immunogenic epitopes." Two prominent immunogenic epitopes were specified by peptides corresponding to sequences spanning R107-120 and R186-199, whereas peptides corresponding to residues R35-50 and R91-106 were only bound in low titer. Three peptides as thyroglobulin conjugates elicited antisera in rabbits that bound intact pertussis toxin by enzyme-linked immunosorbent assay and immunoblot. These peptides were designated "antigenic epitopes." The most prominent antigenic determinant was localized to the N-terminal end of the S2 sequence encompassing residue R1-7. Peptides R35-50 and R91-106 represented two minor antigenic epitopes. Antisera to two additional peptides corresponding to residues R134-149 and R186-199 recognized the S2 subunit only by Western blotting (immunoblotting). Only antiserum raised against peptide R91-106 also recognized the S3 subunit by Western blotting, indicating a marked antigenic and probably also structural difference between the two highly homologous subunits.     

A novel hepatitis B virus variant S 129 (Gln-->Leu): lack of correlation between antigenicity and immunogenicity.

A point mutation has been detected in the "a" determinant of hepatitis B surface antigen (HBsAg) in an infant immunised with hepatitis B vaccine after exposure to hepatitis B virus (HBV). This A-to-T point mutation at nucleotide 540 resulted in a glutamine-to-leucine substitution at amino acid residue 129 (129L). The S gene fragment (nucleotide 58-1072) of this isolate was cloned and used to substitute the wild-type S gene in a plasmid (p3.8II), containing 1.2 copy of full-length HBV genome with expression and replication efficiency. This plasmid p3.8II-129L was used to transfect HepG2 cells. HBsAg expressed by p3.8II-129L showed higher binding efficiency compared with the original plasmid containing the wild-type clone. A panel of 24 anti-HBs monoclonal antibodies (MAbs) was used to characterise the binding efficiency of HBsAg expressed by p3.8II-129L. Eighteen showed higher binding to the antigen, whereas HBsAg expressed by p3.8II-145R gave a consistently lower absorbance or were negative. Surprisingly, when the immunogenicity of plasmid constructs was used for DNA immunisation in Balb/c mice, the anti-HBs response induced by p3.8II-129L was significantly lower than that of the wild-type p3.8II. The lack of correlation between the antigenicity profile (binding of expressed HBsAg to anti-HBs in vitro), and the immunogenicity (induction of anti-HBs by plasmid DNA in vivo) of HBsAg with leucine substitution at position 129 indicates that biological characteristics other than the binding efficiency of HBsAg to anti-HBs could occur in HBsAg variants. These different aspects of the biological characteristics of S mutants merit further investigation.     

Infections by hepatitis B surface antigen gene mutants in Europe and North America

Hepatitis B virus (HBV) mutants have usually been studied in patients in Asia because of the wider use of HBV immunization there and the resultant emergence of viral mutants. Nevertheless, HBV surface antigen (S) gene mutants also are found in Europe and North America. In Europe and North America, HBV with mutations in the portion of the S gene coding the "a" determinant of the hepatitis B surface antigen (HBsAg) have been documented in small numbers of infants born to HBV-infected mothers following post-natal HBV vaccine and hepatitis B immune globulin (HBIG) prophylaxis and in many liver transplant recipients who develop HBV re-infection despite HBIG prophylaxis. In some cases, these mutations have included a glycine to arginine substitution at position 145 (G145R), which results in a conformational change and different reactivity to monoclonal antibody reagents than that of the wild-type virus. Mutations in the a determinant (but not G145R) also have been reported in European patients with chronic HBV infection who have not received HBV vaccine or HBIG. However, it appears that such mutations are only responsible for a small proportion of "occult" or "silent" HBV infections, which are characterized by the presence of HBV DNA in serum in the absence of detectable HBsAg. However, some of these mutant forms of HBV in cases of occult HBV may theoretically escape detection and could present a risk to blood safety.