恶性疟原虫融合蛋白PfCP-5的构建及其免疫原性研究

发布时间：2018-05-11 06:33

本文选题：恶性疟原虫 + 融合蛋白　；参考：《第二军医大学》2007年硕士论文

【摘要】： 疟疾目前仍是一种危害严重的虫媒传染病。全球每年约有3-5亿疟疾病例,其中约300万病人死于恶性疟,大多为五岁以下的儿童。随着疟原虫对抗疟药物以及蚊媒对杀虫剂抗性的产生和扩散,疟疾防治面临很大的困难,因此研究和开发有效的疟疾疫苗已成为防治疟疾潜在的新的途径。疟原虫子孢子通过疟蚊叮咬进入人体后,并侵入肝细胞发育繁殖,产生裂殖子释放入血,并侵入红细胞生长繁殖。阻断疟原虫裂殖子入侵红细胞是抑制红内期原虫生长的重要环节,也是红内期疫苗的主要靶点。研究表明,裂殖子入侵红细胞是由受体和配体介导,位于裂殖子表面的蛋白可能参与该入侵过程,是疫苗研究潜在的靶抗原。本研究旨在研究恶性疟原虫裂殖子表面蛋白(Merozoite Surface Protein MSP)3、4和5的免疫保护功能。MSP3是裂殖子的一个外分泌蛋白,其免疫作用是通过ADCI机制抑制疟原虫生长。研究表明该蛋白的免疫保护功能位于氨基酸第184-210(MSP3b), 203-230(MSP3c)和211-252位(MSP3d),每个区域至少含有一个B细胞和一个T细胞表位,抗MSP3b的抗体通过ADCI效应机制在体外有效杀灭疟原虫。抗MSP3c和MSP3d抗体在体外有较强的ADCI作用,抑制原虫生长。因此MSP3第184-252位的69个氨基酸残基片段是其保护性免疫的功能片段。MSP4是通过GPI位于裂殖子表面的蛋白质,其C末端含有EGF样结构域。MSP5与MSP4高度同源,并在其C末端含有EGF样结构域。针对这两个蛋白EGF区域的抗体在体外能部分抑制疟原虫生长。本研究将上述3个裂殖子表面蛋白的功能区域,即MSP4 C-末端EGF区域(MSP4D,第204-248位氨基酸片段)、MSP3的69个氨基酸片段和MSP5 C-末端EGF区域(MSP5D,第208-251位氨基酸片段)融合组成一个融合抗原,定名为恶性疟原虫融合蛋白5(Plasmodium falciparum Chimeric Protein 5, PfCP-5)。从GenBank获得3D7株恶性疟原虫各片段的序列,并按以下次序排:MSP4D- MSP3-69-MSP5D。按照毕氏酵母密码子使用频率对PfCP-5基因序列进行重新设计,并将第86位的N→Q排除一个糖基化位点。在基因的两端加上合适的酶切位点XhoI/ EcoRI,以及在3’端加上6个His的TAG以便后续蛋白的纯化。人工全合成PfCP-5基因。将合成基因插入过渡载体pPIC9,从而将目的基因前端加上分泌表达的信号肽序列,进而转到表达载体pPIC9K,酶切线性化后电转毕氏酵母。挑取经G418筛选的克隆进行甲醇诱导表达。Western blotting鉴定目的蛋白的表达。采用15升罐进行发酵表达,用Ni-NTA亲和纯化,获得较高纯度的目的蛋白用于后续的动物免疫实验。此外,为纯化MSP3特异的抗体,我们在大肠杆菌中表达了GST-MSP3-69融合蛋白,并分离纯化了该蛋白。动物免疫实验分为ISA720佐剂组、弗氏佐剂组、氢氧化铝/CpG1826联合佐剂组、ISA70MVG佐剂组和ISA 206佐剂组。纯化的PfCP-5蛋白与佐剂乳化后免疫6-8周龄的雌性BALB/c小鼠,每个蛋白佐剂组6只,相应的PBS佐剂对照6只,每只每次免疫20μg蛋白,免疫体积为100μl,皮下注射免疫三次,间隔两周,每次免疫后第8天尾静脉取血。免疫血清用酶联免疫吸附试验(ELISA)检测免疫血清的特异抗体滴度,并进行抗体亚型的分析。结果表明:血清抗体滴度在1/200,000-1/765000范围,其中氢氧化铝/CpG联合佐剂组抗体滴度最高为1/76.5×104,其次是弗氏佐剂组抗体滴度为1/55.2×104,再次ISA720佐剂组抗体滴度为1/48.9×104,ISA70MVG和ISA 206组最低,滴度分别在1/25.3×104和1/20.9×104。氢氧化铝/CpG联合佐剂组,弗氏佐剂组和ISA720佐剂组三组抗体滴度之间没有差异(p0.05),但是三个佐剂组依次分别是ISA70MVG组的3.0倍,2.18倍和1.93倍,同时,分别是206佐剂组的3.66倍,2.64倍和2.33倍。为检测免疫血清能否识别疟原虫天然蛋白,我们以体外培养的恶性疟原虫为抗原,采用间接免疫荧光抗体试验(IFA)进行检测。结果表明,PfCP-5抗体能与疟原虫天然蛋白反应。IFA结果显示:氢氧化铝/CpG联合佐剂组、ISA720佐剂组、弗氏佐剂组、ISA70MVG佐剂组和ISA206佐剂组在同是1:100稀释时,氢氧化铝/CpG联合佐剂组、ISA720佐剂组、弗氏佐剂组阳性较强,而ISA70MVG佐剂组和ISA206佐剂组阳性较弱。抗体亚型分析结果显示,IgG1抗体滴度最高达1/106, IgG2a为其次,最后是IgG2b及IgG3,分别为1/1.2×104和1/0.9×104。采用新西兰白兔为接种动物,实验分为ISA720佐剂组、弗氏佐剂组及氢氧化铝/CpG(CpG2007)联合佐剂组。每组家兔3只,每只每次皮下注射100ug蛋白,注射体积为1ml,间隔三周,免疫三次,每次免疫后第8天取血, ELISA检测血清抗体滴度结果显示:其中ISA720佐剂组最高滴度达1/102×104,弗氏佐剂组在1/72.7×104,氢氧化铝与CpG联合免疫组1/25.2×104,三者之间比较存在显著差异(p0.01)。此外,用大肠杆菌表达GST-MSP3-69融合蛋白,经与ISA720佐剂、弗氏佐剂、氢氧化铝/CpG(CpG1826)联合佐剂分别乳化后免疫BALB/c小鼠。三次免疫后血清ELISA检测抗体滴度分别为1/138×104, 1/116×104和1/72.5×104。用恶性疟原虫FCC1/HN株进行体外抑制试验,结果表明抗PfCP-5的免疫血清没有明显抑制疟原虫生长的作用。用纯化的PfCP-5和GST-MSP3-69为抗原,制备Sepherose 4B-PfCP-5和Sepherose 4B-GST-MSP3-69亲和柱进行特异抗体的分离。用亲和纯化的特异抗体进行体外抑制试验,结果表明在纯化兔抗IgG在终浓度为1.25mg/ml时没有明显抑制原虫生长作用。然而,在单核细胞存在的情况下,抗PfCP-5 IgG终浓度为1.25mg/ml时,抗PfCP-5 IgG和抗GST-MSP3-69 IgG抑制率分别在68%和51.5%。综上所述:本研究构建了PfCP-5融合基因并在毕氏酵母真核表达系统中产生融合蛋白PfCP-5;构建并表达了MSP3(184-252)片段和GST-MSP3-69融合抗原。动物免疫实验表明,这些抗原具有较强的免疫原性,对不同佐剂免疫实验表明在小鼠体内氢氧化铝/CpG(CpG2007)联合佐剂能诱导较强的免疫应答反应。不同佐剂组免疫小鼠血清均能识别体外培养的疟原虫,表明诱导产生针对天然构象表位的抗体。在没有单核细胞存在情况下,抗PfCP-5特异IgG和抗GST-MSP3-69特异IgG没有明显抑制疟原虫生长的作用,但在单核细胞存在的情况下,抑制率分别在68%和51.5%。本试验为后续实验纯化疟疾流行区针对PfCP-5人抗IgG进行该蛋白的功能评价及将PfCP-5或MSP3-69插入本实验室已经构建的疟疾候选疫苗PfCP-2.9中以提高其免疫保护效力提供实验依据。
[Abstract]:Malaria is still a serious insect borne disease. There are about 3-5 billion cases of malaria in the world each year, of which about 3 million patients die of falciparum malaria, most of them are children under five years of age. The effective malaria vaccine has become a potential new way to control malaria.
The Plasmodium spore, which enters the human body through the mosquito bites, invades the liver cells to develop and propagate, produces the merozoites released into the blood and invades the red blood cells to grow and propagate. Blocking the malaria parasite to invade the red blood cells is an important link to inhibit the growth of the protozoa in the red period, and is the main target of the epidemic in the red period. The study shows that the merozoites invade red fine. Cells are mediated by receptors and ligands. Proteins on the surface of the merozoites may be involved in the invasion process and are potential target antigens in vaccine studies. The aim of this study is to study the immunological function of Merozoite Surface Protein MSP (Protein MSP), 3,4 and.MSP3, as an exocrine protein of the merozoite. Its immune function is The ADCI mechanism inhibits the growth of malaria parasites. The study shows that the immune protection function of the protein is located in amino acid 184-210 (MSP3b), 203-230 (MSP3c) and 211-252 (MSP3d), each region contains at least one B cell and one T cell epitopes. The anti MSP3b antibodies kill the Plasmodium effectively in vitro through the ADCI effect mechanism. Resistance to MSP3c and MSP3d is resistant to the Plasmodium. The body has a strong ADCI effect in vitro and inhibits the growth of protozoa. Therefore, the 69 amino acid residues of the 184-252 position of MSP3 are the functional fragment of its protective immunity,.MSP4 is a protein located on the surface of the merozoite by GPI, and its C terminal contains the EGF like domain.MSP5 with MSP4, and contains the EGF like domain at its C terminal. Antibodies to the protein EGF region partially inhibit the growth of Plasmodium falciparum.
In this study, the functional regions of the 3 merozoite surface proteins, namely, the MSP4 C- terminal EGF region (MSP4D, the 204-248 bit amino acid fragment), the 69 amino acid fragments of MSP3 and the MSP5 C- terminal EGF region (MSP5D, the 208-251 bit amino acid fragment) are fused to form a fusion antigen, which is named as the fusion protein 5 of Plasmodium falciparum (Plasmodium falciparum). Chimeric Protein 5, PfCP-5). The sequence of each fragment of Plasmodium falciparum in 3D7 strain was obtained from GenBank and arranged in the following order: MSP4D- MSP3-69-MSP5D. redesigned the sequence of PfCP-5 gene according to the use frequency of Pichia's cipher codon and eliminated a glycosylation site of the eighty-sixth bit N to Q. The appropriate enzyme digestion was added to the two ends of the gene. Point XhoI/ EcoRI, and add 6 His TAG at the 3 'end to purify the subsequent protein. Synthetic PfCP-5 gene is synthesized artificially. The synthetic gene is inserted into the transition carrier pPIC9, and the target gene front end is added to the secretory signal peptide sequence and then transferred to the expression vector pPIC9K. After the enzyme is linearized, it is selected by G418 screening. The expression of the target protein was identified by the cloning of.Western blotting by methanol induction. The protein was expressed in 15 litres and purified with Ni-NTA affinity. The purified target protein was used for subsequent animal immunization experiments. In addition, in order to purify the specific antibody of MSP3, we expressed the GST-MSP3-69 fusion protein in the bacillus Enterobacter and separated it. The protein was purified.
The animal immunization experiment was divided into ISA720 adjuvant group, Freund's adjuvant group, aluminum hydroxide /CpG1826 combined adjuvant group, ISA70MVG adjuvant group and ISA 206 adjuvant group. The purified PfCP-5 protein and the adjuvant were emulsified for 6-8 weeks old female BALB/c mice, each of the protein adjuvant group was 6, and the corresponding PBS adjuvant was 6, each immunized with 20 u g protein and immune body each time. The product was 100 mu L, subcutaneous injection of immunization three times, interval two weeks, eighth days after the immunization of the tail vein to take blood. The immune sera was tested by enzyme linked immunosorbent assay (ELISA) to detect the specific antibody titer of the immune sera and analyze the antibody subtypes. The results showed that the titer of serum antibody was in the range of 1 /200000-1/765000, in which the combined adjuvant of aluminum hydroxide and /CpG was used. The highest titer of the group antibody was 1/76.5 x 104, followed by the antibody titer of the Freund adjuvant group was 1/55.2 x 104, the antibody titer of the second ISA720 adjuvant group was 1/48.9 x 104, and the ISA70MVG and ISA 206 groups were the lowest. The titers were respectively in the 1/25.3 x 104 and 1/20.9 x 104. aluminum hydroxide /CpG combined adjuvant group, and the three groups of antibody titers in the Freund and ISA720 adjuvant group were not between the antibody titers. The difference (P0.05), but the three adjuvant groups were 3 times, 2.18 times and 1.93 times, respectively, 3.66 times, 2.64 times and 2.33 times of the 206 adjuvant group, respectively, to detect the identification of the natural protein of the malaria parasite by the immune sera. We used the indirect immunofluorescence antibody test (IFA) for the antigen of the Plasmodium falciparum in vitro. The results showed that the results showed that PfCP-5 antibody could react with the natural protein of Plasmodium,.IFA results showed that the joint adjuvant group of aluminum hydroxide /CpG, ISA720 adjuvant group, Freund's adjuvant group, ISA70MVG adjuvant group and ISA206 adjuvant group were the same as 1:100 dilution, the aluminum hydroxide /CpG combined adjuvant group, the ISA720 adjuvant group and the Freund adjuvant group were positive, and the ISA70MVG adjuvant was more positive. The group and the ISA206 adjuvant group were weak positive. The antibody subtype analysis showed that the highest titer of IgG1 antibody was 1/106, IgG2a was the second, and the last was IgG2b and IgG3, 1/1.2 x 104 and 1/0.9 x 104., respectively.
The rabbits were inoculated with New Zealand white rabbits. The experiment was divided into ISA720 adjuvant group, Freund's adjuvant group and aluminum hydroxide /CpG (CpG2007) combined adjuvant group. 3 rabbits in each group were injected subcutaneous 100ug protein each time, the volume of the injection was 1ml, the interval was three weeks, the immunization was three times, and the serum antibody titer results showed that the serum antibody titer showed that: The highest titer of the medium ISA720 adjuvant group was 1/102 x 104, the Freund adjuvant group was 1/72.7 x 104, the combined immune group of aluminum hydroxide and CpG was 1/25.2 x 104, and there was a significant difference between the three groups (P0.01). In addition, the GST-MSP3-69 fusion protein was expressed in Escherichia coli, after emulsifying with the combined adjuvant of ISA720 adjuvant, Freund's adjuvant and aluminum hydroxide /CpG (CpG1826), respectively. BALB/c mice were immunized. The titer of serum ELISA after three times of immunization was 1/138 * 104, 1/116 * 104 and 1/72.5 * 104. respectively.
In vitro inhibition test of Plasmodium falciparum FCC1/HN strain showed that anti PfCP-5 immune sera did not significantly inhibit the growth of Plasmodium. Purified PfCP-5 and GST-MSP3-69 were used as antigens to prepare Sepherose 4B-PfCP-5 and Sepherose 4B-GST-MSP3-69 affinity columns for specific anti body isolation. In vitro inhibition test, the results showed that the purified Rabbit anti IgG at the final concentration of 1.25mg/ml did not significantly inhibit the growth of the protozoa. However, in the presence of mononuclear cells, the anti PfCP-5 IgG and the anti GST-MSP3-69 IgG inhibition rates were 68% and 51.5%. respectively when the final concentration of anti PfCP-5 IgG was 1.25mg/ml.
In summary, the PfCP-5 fusion gene was constructed and the fusion protein PfCP-5 was produced in the eukaryotic expression system of Pichia Pichia. The MSP3 (184-252) fragment and GST-MSP3-69 fusion antigen were constructed and expressed. The animal immunization experiments showed that these antigens had strong immunogenicity, and the immunogenicity of different adjuvant immunization experiments showed that the hydrogen oxidation in mice was in vivo. The combined adjuvant of aluminum /CpG (CpG2007) can induce a strong immune response. The mice immunized with different adjuvant groups can identify the Plasmodium in vitro, indicating the induction of antibodies against the natural conformation epitopes. In the absence of mononuclear cells, the anti PfCP-5 specific IgG and the anti GST-MSP3-69 specific IgG did not significantly inhibit the Plasmodium parasite. But in the presence of mononuclear cells, the inhibition rate in the 68% and 51.5%. tests for subsequent experimental purification of malaria epidemic areas for PfCP-5 human resistance to IgG and the insertion of PfCP-5 or MSP3-69 into the malaria candidate vaccine established in our laboratory to improve their immune protection effectiveness are provided. The basis of the test.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383

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