The roles of the glycosylphosphatidylinositol anchor on the production and immunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus expression system

Malarial ookinetes express an immunodominant surface protein (P28) that is a priority candidate for the development of transmission-blocking vaccines. The full length P28 gene from Plasmodium berghei [Pbs21(1-213)] and a deletion construct [Pbs21(1-188)] encoding a protein that lacks the 25 C-terminal amino acids, including the glycosylphosphatidylinositol (GPI) anchor signal, were expressed in insect cells using baculovirus vectors. Pbs21(1-213) protein is strongly hydrophobic, found in the cytoplasm and on the surface of Spodoptera Sf21 cells, and in the culture medium. Pbs21(1-188) protein was largely found in the aqueous phase of the medium and in the cytoplasm of Sf21 cells, but was not detected on the cell surface. The presence of 25 C-terminal amino acids is therefore critical to the attachment of recombinant Pbs21 to the parasite plasma membrane. Mice were immunized subcutaneously or intramuscularly with affinity purified recombinant Pbs21(1-213), Pbs21(1-188) or native Pbs21 proteins. Following two immunizations, native Pbs21 induces higher titres when administered by either route, than the recombinant protein bearing an insect GPI anchor, which in turn is markedly more immunogenic than the recombinant polypeptide lacking a GPI anchor. When specific anti Pbs21 antibody titres exceeded 1 mg/ml all three antigens were capable of inducing transmission blockade > or = 90%, below 1 mg/ml blockade did not correlate with antibody concentration.     

Ookinete antigens of Plasmodium berghei. Appearance on the zygote surface of an Mr 21 kD determinant identified by transmission-blocking monoclonal antibodies

Zygotes and ookinetes of the rodent malaria Plasmodium berghei can be enriched 50-fold, from whole blood cultures by ammonium chloride lysis. Three monoclonal antibodies (MoAbs) raised against such enriched preparations specifically bind to a determinant of Mr 21 kD as assessed by 125I-labelled goat anti-mouse IgG probed immunoblots of Western transfers of SDS-PAGE gels. Indirect immunofluorescence indicates that the 21 kD determinant bound by specific MoAbs, whilst not detectable on gametocytes or gametes, appears on the parasite surface within 2 h of exflagellation/fertilization and increases thereafter. The three MoAbs specifically binding the 21 kD determinant block oocyst development in mosquitoes by at least 90%, as assessed either by in-vitro membrane feeds or by live feeds on passively immunized mice. These MoAbs reduce ookinete formation in vitro by between 52 and 100%. Possible mechanisms of action of these MoAbs are discussed.     

Studies on the immunogenicity of a recombinant ookinete surface antigen Pbs21 from Plasmodium berghei expressed in Escherichia coli

Plasmodium berghei ookinete surface antigen (Pbs21), was produced as a fusion product with maltose binding protein (MBP) in Escherichia coli and used to induce transmission-blocking immunity in mice. Specificity of induced antibody was confirmed by Western blotting with native ookinete Pbs21, and by the indirect immunofluorescent antibody test on ookinete bloodfilms. Immunized mice were infected with P. berghei and transmission to Anopheles stephensi mosquitoes determined by both the intensity and prevalence of oocyst infections. Compared with a control group immunized with MBP alone the maximum blockade of oocyst intensity was 66% in the mice immunized with recombinant MBP-Pbs21. Over nine experiments blockade averaged only 33%. By comparison with native Pbs21 protein, which usually induces 90% blockade, our data suggests the recombinant protein produced in this bacterial system is a less effective immunogen despite expressing epitopes recognized by known transmission-blocking monoclonal antibodies.     

Three non-repeated transmission blocking epitopes recognized in the 21 kD surface antigen of zygotes-ookinetes of Plasmodium berghei.

Two-site and competitive ELISA have been developed against a surface antigen of zygotes-ookinetes of Plasmodium berghei using monoclonal antibodies (MoAbs) which block transmission of the parasite to the mosquito. Three such MoAbs have been studied, each of which recognized a protein of an Mr 21 kD (Pbs21) using immunoblot techniques. The assays showed that there are at least 3 single B-cell epitopes expressed in Pbs21. One epitope recognized by MoAb 17.9 is conformation dependent and antibodies bound to it interfered with other MoAbs (12.1 and 13.1) each recognizing a different, apparently linear epitope. Glycosylation might not be relevant to the binding of any of the antibodies tested to their respective epitopes.     

Three non-repeated transmission blocking epitopes recognized in the 21 kD surface antigen of zygotes-ookinetes of Plasmodium berghei

Summary Two-site and competitive ELISA have been developed against a surface antigen of zygotes-ookinetes of Plasmodium berghei using monoclonal antibodies (MoAbs) which block transmission of the parasite to the mosquito. Three such MoAbs have been studied, each of which recognized a protein of an M_r 21 kD (Pbs21) using immunoblot techniques. The assays showed that there are at least 3 single B-cell epitopes expressed in Pbs21. One epitope recognized by MoAb 17.9 is conformation dependent and antibodies bound to it interfered with other MoAbs (12.1 and 13.1) each recognizing a different, apparently linear epitope. Glycosylation might not be relevant to the binding of any of the antibodies tested to their respective epitopes.     

Interferon-gamma and the induction of protective lgG2a antibodies in non-lethal Plasmodium berghei infections of mice

Mice treated with anti-IFN-gamma monoclonal antibodies were unable to recover from infection with an attenuated variant of P. berghei (Pb XAT) which causes non-lethal malaria in normal mice. On the other hand, treatment with anti-lL-4 monoclonal antibodies had no effect on the course of infection. IFN-gamma was produced by spleen cells in vitro during the early phase of the infection. Treatment with anti-IFN-gamma suppressed development of an anti-plasmodial IgG2a immunoglobulin isotype in the serum of infected mice whereas anti-IL-4 interfered with the IgGl response. An lgG2a fraction of immune serum collected from mice that had recovered from Pb XA T transferred immunity to naive mice but the IgGl fraction did not. When glutaraldehyde fixed parasitized erythrocytes were incubated with immune serum in suspension, specific IgG2a antibodies were detected by fluorescein staining on the membranes of cells infected with mature stages of parasites. These results indicate that IFN-gamma is a key to inducing B cells to produce the protective anti-plasmodial IgG2a immunoglobulin isotype. Antibody-dependent cell-mediated parasite killing seems to be involved in the mechanism of recovery from infection with Pb XAT.     

Protective and pathological activity in serum of mice developing resistance to Plasmodium berghei infection

Summary The serum from mice developing resistance against Plasmodium berghei infection using chemotherapeutic treatment has been analysed in vivo and in vitro. During the immunization period pathological as well as protective activities which could be transferred by serum were generated. The pathological activity, which was defined as destruction of erythrocytes in normal recipient mice, was generated early in the immunization procedure, peaked at day 21, and decreased to undetectable levels by day 35. After reinfection of the donor mice the pathological activity reappeared in the serum, and was maintained for at least 56 days. Analysis of the transferred serum samples showed the presence of anti-erythrocyte antibodies (ELISA), but no correlation with the in-vivo anti-erythrocyte effect could be found. The anti-erythrocyte effect of the serum samples indirectly increased the parasitaemia in the recipient mice through the induction of reticulocytosis. The protective effect of the serum samples could only be detected in samples taken from animals beyond day 61 of the immunization procedure. This net protective effect was reflected in a decreased parasitaemia at 7 days after challenge of the recipient mice with P. berghei infected erythrocytes. The protective activity of the serum was correlated with high titres of anti-erythrocyte antibodies. Anti-erythrocyte antibody titres were strongly correlated with titres against heterologous red blood cells as well as total immunoglobulin content of the serum samples, indicative of polyclonal activation of lymphocytes. Except for IgGl, all (sub-) classes were elevated during the immunization procedure, of which IgG3 was abundant. After immunity was obtained these immunoglobulin levels remained high, and the relative amount of IgGl in the serum was restored.     

Specific lgG1 and lgG2 antibody responses of dogs to Leishmania infantum and other parasites

Sera from dogs naturally infected with Leishmania infantum were analysed for the IgG subclass specificity of their antibody response by ELISA. Dogs infected with L. infantum produced both IgGl and IgG2 antibodies with IgG2 being associated with asymptomatic infections and IgGl being associated with disease (symptomatic dogs, non- or low-responsive to chemotherapy). The differential responses of IgG] and IgGl serum antibodies in asymptomatic and symptomatic dogs may indicate a dichotomous immune response to infection with L. infantum. To confirm this, on a broader scale, sera from dogs naturally exposed to an asymptomatic protozoan infection, Toxoplasma gondii, were also analysed as were sera from dogs exposed to the helminths, Dirofilaria immitis and Toxocara canis. Antibodies specific for T. gondii antigen detected in sera from 17 dogs were of the IgG2 subclass only. Both IgGl and IgG2 antibodies to D. immitis andl. canis were present in the sera of naturally infected dogs but IgGl appeared to be the predominant subclass. Furthermore, in dogs experimentally infected with T. canis, selective regulation ofIgG2 and IgGl responses was apparent since production of the two subclasses occurred at different times following infection, with IgGl levels declining as IgGl levels rose. Thus, the analysis of IgG subsets in parasitized dogs provides evidence of a dichotomous response to infection: IgGl is associated with asymptomatic protozoan infections and IgGl is associated with helminth infections and disease caused by protozoan infection.     

Impaired immune responsiveness in Plasmodium berghei immune mice

Mice immunized against Plasmodium berghei parasites by drug-controlled infection exhibited decreased immunoresponsiveness against rabbit red blood cells (RRBC). Increasing RRBC antigen dose increased responsiveness, but agglutinating anti-RRBC antibodies of the IgG class remained undetectable. Clearance of colloidal carbon from the bloodstream of malaria-immunized mice was not different from controls. Removal of all the persistent parasites from immune mice did not restore responsiveness until 140 days after treatment, suggesting that the parasite per se did not influence responsiveness directly. Because of this, and because of the fact that priming of mice with RRBC before P. berghei immunization was not more effective than priming after immunization, it was concluded that antigen uptake and subsequent presentation were not impaired in P. berghei immune mice, in contrast to infected mice. Anti-RRBC antibodies were detected in serum of P. berghei immune mice, but regulation of responsiveness to RRBC by transfer of such immune mouse serum was not found. Immunoglobulin levels, especially of the IgG2 and IgG3 subclass were elevated in sera of P. berghei immune mice, which indicated an LPS-like polyclonal activation. The results also suggest that during drug-controlled infection, which leads to immunity against infection, a state of B-cell tolerance is induced.     

免疫佐剂增强乙型肝炎核心抗原核酸疫苗免疫应答的小鼠初步实验

目的 观察小鼠实验中免疫佐剂QS－21对乙型肝炎核心抗原（HBcAg)核酸疫苗免疫应答的增强作用。方法 基因枪轰击法接种HBcAg核酸疫苗或对照质粒,皮内注射法给予QS－21;间接酶联免疫吸附试验（ELISA）法检测小鼠血清抗－HBc(IgG及IgG亚类）水平;^51 铬释 放法检 小鼠脾 细胞特异性CTL杀伤活性。结果 Balb/c和C57BL／6小鼠单用疫苗组和疫苗加QS－21组的血清抗－HBc 终点滴度均分别为1:328050和1:984150;两组的-HBc-I gG亚类均以IgG2a略占优势;当效：靶比为12：1时,Balb/c小鼠单用疫苗组和疫苗加QS－21组的特异性CTL杀伤活性分别为51.1%和63.6%;C57BL／6小鼠两组的CTL杀伤活性分别为53.1%和68.1%。结论 QS－21对HBcAg核酸疫苗所诱导的小鼠特异性抗体及CTL应答均有增强作用。     

Antibody suppression in mice infected with Nippostrongylus brasiliensis

Summary A/SN mice infected with N. brasiliensis showed depressed anti-DNP antibody responses following immunization with DNP-Asc in alum. The immunosuppression was only observed when infection preceded immunization by between 2 and 7 days, and was not achieved when the interval was extended to 10 days. The suppression lasted at least 50 days, and affected IgE levels more than IgGl or IgG agglutinating anti-DNP antibodies. A high dose of infective larvae (500–1000 per mouse) was necessary to induce suppression. Use of low dose irradiation indicated a parasite-induced radiosensitive component of the mouse immune system which negatively regulated the anti-DNP IgE response. These results suggested that the parasite could induce suppression in an analogous manner to sequential antigen-induced suppression (AIS).     

Cross-protection between attenuated Plasmodium berghei and P. yoelii sporozoites

An attenuated Plasmodium falciparum sporozoite (PfSPZ) vaccine is under development, in part, based on studies in mice with P. berghei. We used P. berghei and P. yoelii to study vaccine-induced protection against challenge with a species of parasite different from the immunizing parasite in BALB/c mice. One-hundred percent of mice were protected against homologous challenge. Seventy-nine percent immunized with attenuated P. berghei sporozoite (PbSPZ) (six experiments) were protected against challenge with P. yoelii sporozoite (PySPZ), and 63% immunized with attenuated PySPZ (three experiments) were protected against challenge with PbSPZ. Antibodies in sera of immunized mice only recognized homologous sporozoites and could not have mediated protection against heterologous challenge. Immunization with attenuated PySPZ or PbSPZ induced CD8+ T cell-dependent protection against heterologous challenge. Immunization with attenuated PySPZ induced CD8+ T cell-dependent protection against homologous challenge. However, homologous protection induced by attenuated PbSPZ was not dependent on CD8+ or CD4+ T cells, and depletion of both populations only reduced protection by 36%. Immunization of C57BL/10 mice with PbSPZ induced CD8+ T cell-dependent protection against P. berghei, but no protection against P. yoelii. The cross-protection data in BALB/c mice support testing a human vaccine based on attenuated PfSPZ for its efficacy against P. vivax.     

广州管圆线虫半胱氨酸蛋白酶抑制剂基因的克隆表达及免疫保护作用研究

目的对广州管圆线虫半胱氨酸蛋白酶抑制剂(cystatin)基因进行克隆、原核表达,并对表达产物的免疫保护作用作出初步鉴定。方法将广州管圆线虫cystatin基因克隆入载体pGEX-4T-1中,转化大肠杆菌BL21进行诱导表达。用纯化融合蛋白隔周免疫小鼠1次,共3次,设GST免疫及未免疫对照组,末次免疫后1w用广州管圆线虫Ⅲ期幼虫攻击感染,3w后剖杀小鼠,计算减虫率及保护率,并测定小鼠特异性IgG抗体水平变化。结果成功构建了pGEX-4T-1/cystatin重组表达质粒,并在大肠杆菌中得以高效可溶性表达,融合蛋白免疫小鼠后诱导产生了58.1%的减虫率及20%的保护率,与对照组相比减虫效果显著(P<0.01)。结论广州管圆线虫cystatin基因能在大肠杆菌中高效表达并可诱导小鼠产生一定水平的保护性免疫力。     

阴道毛滴虫单克隆抗体的制备和鉴定

用阴道毛滴虫滋养体免疫BALB/c小鼠,取脾细胞与NS_1鼠骨髓瘤细胞融合,选择4株(2A2,2A4,2A12,2H9)杂交瘤细胞,证明能分泌效价高、特异性强的单克隆抗体(McAb)。亚类鉴定为IgG1(2A2,2A4),IgG2b(2A12),IgG3(2H9)。2H9株和其他3株McAb分别能抗阴道毛滴虫细胞核和细胞膜。除2H9株外均可凝集或杀伤虫体,但2A2、2A4和2A12株介导的细胞毒性分别是不依赖和依赖补体的。这4株McAb与杜氏利什曼原虫前鞭毛体、枯氏锥虫锥鞭毛体和蓝氏贾第鞭毛虫滋养体无交叉反应。     

Structural and molecular specificity of antibody responses in mice immune to third stage larvae of Onchocerca volvulus

Immunization of mice with irradiated Onchocerca volvulus infective stage larvae (L3) has been demonstrated to confer protection against challenge infections with these larvae. Additionally, cytokine level measurements and cytokine depletion studies have shown that both IL-4 and IL-5 are important in generating a protective immune response against O. volvulus challenge infections, thus suggesting a dependency of protective immunity on IgG1, IgE and/or eosinophils. In the present study, we examined the humoral responses of immunized mice to O. volvulus L3 antigens. ELISA measurements of total serum antibody levels indicated that IgE was the only antibody isotype elevated in mice immunized with O. volvulus L3. IgM from immunized mice was the only isotype that recognized surface antigens on intact O. volvulus L3. IgG1, IgG3, IgE and IgA recognized internal parasite antigens on O. volvulus L3 frozen sections. Western blot analysis of L3 proteins showed that in serum from mice immunized with O. volvulus L3 IgG1, IgG2a/2b, IgA, and IgE, as well as IgM, recognized unique L3 proteins. Antibodies in serum from L3 immunized mice were able to detect O. volvulus adult antigens in a pattern similar to the recognition found in O. volvulus L3. Some L3 antigens were shared by adults, while other antigens were L3 specific. The ELISA, immunohistochemistry and Western blot findings thus demonstrate a complex pattern of antigen recognition of parasite antigens by antibodies found in mice immune to the L3 of O. volvulus     

MHC-restricted antibody responses to Trichuris muris excretory/secretory (E/S) antigen

Summary Two panels of H-2 recombinant mice were used in a detailed serological study to analyse the role of H-2-linked genes in the control of the antibody response to excretory/secretory (E/S) antigens of Trichuris muris. An apparent H-2 ^q ( 1-A ^q) restriction on the early development of high levels of IgGl antibody to E/S antigen was revealed by ELISA. No such restriction was demonstrated for the specific IgG2a response patterns. Recognition of two high molecular weight antigens (90–95 kDa, 105–110 kDa) by IgG antibodies was also shown to be almost exclusively H-2 ^q restricted and may be related at least in part to the high antibody levels seen for H-2 ^q strains of mice. Immune serum from resistant (B10.BR × B10.G) Fl hybrid mice ( H-2 ^qk) containing high levels of IgGl antibodies specific for T. muris E/S and IgG antibodies which recognized the 90–95 kDa and 105–110 kDa E/S antigens was effective in transferring protection to the non-responsive B10.BR mouse strain as seen on day 35 post-infection (p.i.). It is suggested that the IgG responses described for the generally very resistant H-2 ^q mouse strains may contribute to, but not be an absolute requirement for. protective immunity, antibody-mediated damage facilitating a subsequent cellular attack in certain strains of mice.     

The role of CD4 cells in protective immunity to Brugia pahangi

The BALB/c mouse immunized sub-cutaneously (s.c.) with 45 kRad attenuated third stage larvae (L₃) of the lymphatic filarial nematode Brugia pahangi is strongly immune to a challenge infection (75–100% reduction in recovery at day six post challenge). Analysis of spleen cell supernatants from immunized mice re-stimulated in vitro, with parasite antigen or the non specific T cell mitogen Con-A reveals a cytokine profile (IL-4, IL-5 and IL-9) which indicates that the Th2 subset of CD4 cells has been expanded. In an attempt to formally prove a critical role for CD4 cells in immunity in this model system, immunized mice were given either anti-CD4 or anti-CD8 neutralizing antibodies. Administration of anti-CD4 antibody had a significant and detrimental effect on the immune response whereas anti-CD8 antibody had a negligible effect on immunity. The efficacy of antibody in neutralizing their target cells was determined by fluorescence activated cell sorting analysis (FACS). Spleen cells from anti-CD4 treated immunized mice, when re-stimulated with parasite antigen had a significantly reduced potential to secrete IL-4, IL-5 and IL-9 in vitro and serum from these mice had reduced levels of parasite specific IgG and IgE. These results demonstrate a critical role for CD4 T cells in host protective immunity to B. pahangi in vivo and strongly suggest that some component of the Th2 response plays an important role in the immune response elicited in this model system.     

Anti-idiotypic antibodies function as a surrogate surface epitope of Brugia malayi infective larvae.

Anti-idiotypic (AB2) antibodies were generated in rabbits following immunization with a murine IgM monoclonal antibody (AB1) recognizing a surface determinant of Brugia malayi infective stage larvae. AB2 specifically inhibited the binding of AB1 to B. malayi larvae. Furthermore, AB2 had the ability to mimic the original antigen since mice immunized with AB2 possessed serum antibodies (AB3) specific for the B. malayi surface determinant. The presence of anti-surface antibodies (AB3 and AB1) induced either by AB2 immunization or by administration of AB1, did not alter the outcome of an intraperitoneal infection of B. malayi larvae in BABL/c mice when compared to untreated animals. AB3 antibodies like AB1, were IgM, thus indicating an isotype restricted response to the B. malayi epitope. There were no detectable cell mediated responses to the surface determinant in mice immunized with AB2, assessed by lymphocyte blastogenesis or IL3 production in vitro in response to the idiotope as presented by living larvae. The lack of cellular responses and/or the previously demonstrated rapid shedding of the epitope may explain the inability of AB1 or AB2 to protect mice against larval challenge in this study.     

伯氏疟原虫顶端膜抗原1胞外区及其亚区的表达与免疫保护作用分析

目的 表达伯氏疟原虫顶端膜抗原1（PbAMA-1）胞外区E及其各亚区，分析其免疫原性和免疫保护作用。 方法 抽提伯氏疟原虫基因组，对PbAMA-1基因测序，依据该序列采用毕赤酵母密码子使用频率重新设计PbA-MA-1基因序列。将其胞外区E分为3个彼此重叠的基因片段DⅠ、DⅡ和 DⅢ并进行优化，人工合成后在大肠埃希菌中诱导表达。表达产物经纯化和重折叠，分别与福氏佐剂乳化后免疫小鼠和家兔, 均免疫3次， 间隔2周。每次免疫剂量, 小鼠为20μg, 家兔为100 μg。ELISA检测抗体效价，并以疟原虫攻击实验确定各抗原的免疫保护效果。 结果 测得的PbAMA-1基因序列与Sanger测序中心公布的序列完全一致。密码子优化并人工合成的DⅠ、DⅡ、DⅢ 和E目的基因片段在大肠埃希菌表达载体pET32a均获得诱导表达，经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析，各目的基因表达产物大小与预计的相对分子质量Mr 44 300、Mr 33 300、Mr 29 900和Mr 67 200相一致。表达产物经镍离子螯合柱（Ni-NTA柱）纯化和谷胱甘肽(GSH)氧化还原法体外重折叠，蛋白纯度达90%以上, 各纯化的重组蛋白在小鼠体内均激发产生高滴度抗体。其中，完整的胞外区E第3次免疫后抗体滴度为（34.4±0.15）×10^-4，与其他组相比，免疫原性较其他各亚区更强(t=5.66，P<0.01；t=2.27，P<0.05和t=5.05，P<0.01)。取家兔免疫血清与感染伯氏疟原虫ANKA株的小鼠血膜抗原片进行间接荧光抗体试验(IFAT)，均为阳性，其中抗E的抗血清免疫荧光强度最高，与ELISA检测的抗体水平一致。取家兔抗血清对小鼠伯氏疟原虫粗提抗原进行蛋白质印迹（Western blot）分析，产生特异性反应条带，表明能够识别天然的PbAMA-1蛋白。免疫小鼠在以伯氏疟原虫攻击实验中得到部分保护，DⅠ、DⅡ、DⅢ 和E各免疫组小鼠与佐剂对照组相比，存活时间明显延长(t=2.78，P<0.05；t=2.67，P<0.05；t=3.46，P<0.01和t=3.50，P<0.01)。 结论 人工合成的PbAMA-1具有良好的免疫原性和免疫保护作用，完整的胞外区E较其各亚区表现出更好的免疫原性。     

EB病毒潜伏膜蛋白2的B细胞表位免疫原性研究

目的 分析EB病毒(EBV)潜伏膜蛋白2(LMP2)B淋巴细胞表位的免疫原性.方法 利用生物信息学技术筛选出EBV LMP2可能的优势B淋巴细胞表位LMP2199-209、LMP2318-322和LMP2381-391,将其相应基因片段分别克隆至pET32a(+)载体并转化至大肠埃希菌BL21(DE3)菌株诱导表达,表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹分析鉴定,通过Ni-NTA离子螯合亲和层析柱纯化.分别以纯化的表位蛋白作为免疫原,皮下多点注射BALB/c小鼠,设pET32a(+)蛋白和PBS为对照.分别以3个表位蛋白作包被抗原,ELISA法检测第0、3、6周小鼠血清中相应表位特异性抗体IgG,并于免疫后第6周检测各免疫组小鼠血清抗体识别天然EBV抗原的能力.结果 在原核表达体系中分别获得了表位LMP2199-209、LMP2318-322和LMP2381 391融合表达产物.纯化的表位蛋白免疫小鼠,血清中分别能检测到相应的表位特异性抗体IgG,其抗体水平随着免疫次数的增加而呈现逐渐升高的趋势,表位LMP2318-322免疫组第3、6周小鼠血清抗体显著高于pET32a(+)蛋白对照组(F=493.85、773.99,均P＜0.05),LMP2381 391免疫组第3、6周抗体水平亦显著高于pET32a(+)蛋白对照组(F=926.33、309.14,均P＜0.05),而表位LMP2199 209免疫组至第6周特异性抗体高于pET32a(+)蛋白对照组(F=87.27,P＜0.05).3个表位蛋白免疫小鼠血清抗体IgG均能识别EBV天然抗原,以表位LMP2199-209和LMP2381-391免疫组抗EBV-IgG生成水平尤为显著.结论 筛选的EBV LMP2可能的优势B淋巴细胞表位LMP2199-209、LMP2318-322和LMP2381-391通过原核表达体系中制备的表位蛋白,具有较好的免疫原性,可进一步用于EBV感染及其相关肿瘤表位疫苗的研究.