本文選題：惡性瘧原蟲(chóng) + 融合蛋白�。� 參考：《第二軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 瘧疾目前仍是一種危害嚴(yán)重的蟲(chóng)媒傳染病。全球每年約有3-5億瘧疾病例,其中約300萬(wàn)病人死于惡性瘧,大多為五歲以下的兒童。隨著瘧原蟲(chóng)對(duì)抗瘧藥物以及蚊媒對(duì)殺蟲(chóng)劑抗性的產(chǎn)生和擴(kuò)散,瘧疾防治面臨很大的困難,因此研究和開(kāi)發(fā)有效的瘧疾疫苗已成為防治瘧疾潛在的新的途徑。 瘧原蟲(chóng)子孢子通過(guò)瘧蚊叮咬進(jìn)入人體后,并侵入肝細(xì)胞發(fā)育繁殖,產(chǎn)生裂殖子釋放入血,并侵入紅細(xì)胞生長(zhǎng)繁殖。阻斷瘧原蟲(chóng)裂殖子入侵紅細(xì)胞是抑制紅內(nèi)期原蟲(chóng)生長(zhǎng)的重要環(huán)節(jié),也是紅內(nèi)期疫苗的主要靶點(diǎn)。研究表明,裂殖子入侵紅細(xì)胞是由受體和配體介導(dǎo),位于裂殖子表面的蛋白可能參與該入侵過(guò)程,是疫苗研究潛在的靶抗原。本研究旨在研究惡性瘧原蟲(chóng)裂殖子表面蛋白(Merozoite Surface Protein MSP)3、4和5的免疫保護(hù)功能。MSP3是裂殖子的一個(gè)外分泌蛋白,其免疫作用是通過(guò)ADCI機(jī)制抑制瘧原蟲(chóng)生長(zhǎng)。研究表明該蛋白的免疫保護(hù)功能位于氨基酸第184-210(MSP3b), 203-230(MSP3c)和211-252位(MSP3d),每個(gè)區(qū)域至少含有一個(gè)B細(xì)胞和一個(gè)T細(xì)胞表位,抗MSP3b的抗體通過(guò)ADCI效應(yīng)機(jī)制在體外有效殺滅瘧原蟲(chóng)�？筂SP3c和MSP3d抗體在體外有較強(qiáng)的ADCI作用,抑制原蟲(chóng)生長(zhǎng)。因此MSP3第184-252位的69個(gè)氨基酸殘基片段是其保護(hù)性免疫的功能片段。MSP4是通過(guò)GPI位于裂殖子表面的蛋白質(zhì),其C末端含有EGF樣結(jié)構(gòu)域。MSP5與MSP4高度同源,并在其C末端含有EGF樣結(jié)構(gòu)域。針對(duì)這兩個(gè)蛋白EGF區(qū)域的抗體在體外能部分抑制瘧原蟲(chóng)生長(zhǎng)。 本研究將上述3個(gè)裂殖子表面蛋白的功能區(qū)域,即MSP4 C-末端EGF區(qū)域(MSP4D,第204-248位氨基酸片段)、MSP3的69個(gè)氨基酸片段和MSP5 C-末端EGF區(qū)域(MSP5D,第208-251位氨基酸片段)融合組成一個(gè)融合抗原,定名為惡性瘧原蟲(chóng)融合蛋白5(Plasmodium falciparum Chimeric Protein 5, PfCP-5)。從GenBank獲得3D7株惡性瘧原蟲(chóng)各片段的序列,并按以下次序排:MSP4D- MSP3-69-MSP5D。按照畢氏酵母密碼子使用頻率對(duì)PfCP-5基因序列進(jìn)行重新設(shè)計(jì),并將第86位的N→Q排除一個(gè)糖基化位點(diǎn)。在基因的兩端加上合適的酶切位點(diǎn)XhoI/ EcoRI,以及在3’端加上6個(gè)His的TAG以便后續(xù)蛋白的純化。人工全合成PfCP-5基因。將合成基因插入過(guò)渡載體pPIC9,從而將目的基因前端加上分泌表達(dá)的信號(hào)肽序列,進(jìn)而轉(zhuǎn)到表達(dá)載體pPIC9K,酶切線性化后電轉(zhuǎn)畢氏酵母。挑取經(jīng)G418篩選的克隆進(jìn)行甲醇誘導(dǎo)表達(dá)。Western blotting鑒定目的蛋白的表達(dá)。采用15升罐進(jìn)行發(fā)酵表達(dá),用Ni-NTA親和純化,獲得較高純度的目的蛋白用于后續(xù)的動(dòng)物免疫實(shí)驗(yàn)。此外,為純化MSP3特異的抗體,我們?cè)诖竽c桿菌中表達(dá)了GST-MSP3-69融合蛋白,并分離純化了該蛋白。 動(dòng)物免疫實(shí)驗(yàn)分為ISA720佐劑組、弗氏佐劑組、氫氧化鋁/CpG1826聯(lián)合佐劑組、ISA70MVG佐劑組和ISA 206佐劑組。純化的PfCP-5蛋白與佐劑乳化后免疫6-8周齡的雌性BALB/c小鼠,每個(gè)蛋白佐劑組6只,相應(yīng)的PBS佐劑對(duì)照6只,每只每次免疫20μg蛋白,免疫體積為100μl,皮下注射免疫三次,間隔兩周,每次免疫后第8天尾靜脈取血。免疫血清用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)免疫血清的特異抗體滴度,并進(jìn)行抗體亞型的分析。結(jié)果表明:血清抗體滴度在1/200,000-1/765000范圍,其中氫氧化鋁/CpG聯(lián)合佐劑組抗體滴度最高為1/76.5×104,其次是弗氏佐劑組抗體滴度為1/55.2×104,再次ISA720佐劑組抗體滴度為1/48.9×104,ISA70MVG和ISA 206組最低,滴度分別在1/25.3×104和1/20.9×104。氫氧化鋁/CpG聯(lián)合佐劑組,弗氏佐劑組和ISA720佐劑組三組抗體滴度之間沒(méi)有差異(p0.05),但是三個(gè)佐劑組依次分別是ISA70MVG組的3.0倍,2.18倍和1.93倍,同時(shí),分別是206佐劑組的3.66倍,2.64倍和2.33倍。為檢測(cè)免疫血清能否識(shí)別瘧原蟲(chóng)天然蛋白,我們以體外培養(yǎng)的惡性瘧原蟲(chóng)為抗原,采用間接免疫熒光抗體試驗(yàn)(IFA)進(jìn)行檢測(cè)。結(jié)果表明,PfCP-5抗體能與瘧原蟲(chóng)天然蛋白反應(yīng)。IFA結(jié)果顯示:氫氧化鋁/CpG聯(lián)合佐劑組、ISA720佐劑組、弗氏佐劑組、ISA70MVG佐劑組和ISA206佐劑組在同是1:100稀釋時(shí),氫氧化鋁/CpG聯(lián)合佐劑組、ISA720佐劑組、弗氏佐劑組陽(yáng)性較強(qiáng),而ISA70MVG佐劑組和ISA206佐劑組陽(yáng)性較弱�？贵w亞型分析結(jié)果顯示,IgG1抗體滴度最高達(dá)1/106, IgG2a為其次,最后是IgG2b及IgG3,分別為1/1.2×104和1/0.9×104。 采用新西蘭白兔為接種動(dòng)物,實(shí)驗(yàn)分為ISA720佐劑組、弗氏佐劑組及氫氧化鋁/CpG(CpG2007)聯(lián)合佐劑組。每組家兔3只,每只每次皮下注射100ug蛋白,注射體積為1ml,間隔三周,免疫三次,每次免疫后第8天取血, ELISA檢測(cè)血清抗體滴度結(jié)果顯示:其中ISA720佐劑組最高滴度達(dá)1/102×104,弗氏佐劑組在1/72.7×104,氫氧化鋁與CpG聯(lián)合免疫組1/25.2×104,三者之間比較存在顯著差異(p0.01)。此外,用大腸桿菌表達(dá)GST-MSP3-69融合蛋白,經(jīng)與ISA720佐劑、弗氏佐劑、氫氧化鋁/CpG(CpG1826)聯(lián)合佐劑分別乳化后免疫BALB/c小鼠。三次免疫后血清ELISA檢測(cè)抗體滴度分別為1/138×104, 1/116×104和1/72.5×104。 用惡性瘧原蟲(chóng)FCC1/HN株進(jìn)行體外抑制試驗(yàn),結(jié)果表明抗PfCP-5的免疫血清沒(méi)有明顯抑制瘧原蟲(chóng)生長(zhǎng)的作用。用純化的PfCP-5和GST-MSP3-69為抗原,制備Sepherose 4B-PfCP-5和Sepherose 4B-GST-MSP3-69親和柱進(jìn)行特異抗體的分離。用親和純化的特異抗體進(jìn)行體外抑制試驗(yàn),結(jié)果表明在純化兔抗IgG在終濃度為1.25mg/ml時(shí)沒(méi)有明顯抑制原蟲(chóng)生長(zhǎng)作用。然而,在單核細(xì)胞存在的情況下,抗PfCP-5 IgG終濃度為1.25mg/ml時(shí),抗PfCP-5 IgG和抗GST-MSP3-69 IgG抑制率分別在68%和51.5%。 綜上所述:本研究構(gòu)建了PfCP-5融合基因并在畢氏酵母真核表達(dá)系統(tǒng)中產(chǎn)生融合蛋白PfCP-5;構(gòu)建并表達(dá)了MSP3(184-252)片段和GST-MSP3-69融合抗原。動(dòng)物免疫實(shí)驗(yàn)表明,這些抗原具有較強(qiáng)的免疫原性,對(duì)不同佐劑免疫實(shí)驗(yàn)表明在小鼠體內(nèi)氫氧化鋁/CpG(CpG2007)聯(lián)合佐劑能誘導(dǎo)較強(qiáng)的免疫應(yīng)答反應(yīng)。不同佐劑組免疫小鼠血清均能識(shí)別體外培養(yǎng)的瘧原蟲(chóng),表明誘導(dǎo)產(chǎn)生針對(duì)天然構(gòu)象表位的抗體。在沒(méi)有單核細(xì)胞存在情況下,抗PfCP-5特異IgG和抗GST-MSP3-69特異IgG沒(méi)有明顯抑制瘧原蟲(chóng)生長(zhǎng)的作用,但在單核細(xì)胞存在的情況下,抑制率分別在68%和51.5%。本試驗(yàn)為后續(xù)實(shí)驗(yàn)純化瘧疾流行區(qū)針對(duì)PfCP-5人抗IgG進(jìn)行該蛋白的功能評(píng)價(jià)及將PfCP-5或MSP3-69插入本實(shí)驗(yàn)室已經(jīng)構(gòu)建的瘧疾候選疫苗PfCP-2.9中以提高其免疫保護(hù)效力提供實(shí)驗(yàn)依據(jù)。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R383

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