HIV-1膜抗原改造及免疫原性評價
發(fā)布時間：2019-06-24 20:59
【摘要】： 人類免疫缺陷病毒(HIV)的膜蛋白(Env)位于病毒的表面，是中和抗體作用的主要靶位，但野生型Env在機(jī)體免疫壓力下易發(fā)生免疫逃逸，這可能與其復(fù)雜的構(gòu)象和表面寡糖鏈有關(guān)。本課題試圖通過刪除寡糖鏈、復(fù)雜氨基酸側(cè)鏈及高變區(qū)，來暴露目前已知的及CD4i(CD4 induced)區(qū)潛在的中和抗體表位，以期誘導(dǎo)廣譜有效的中和抗體，同時將體液免疫與細(xì)胞免疫結(jié)合起來，力圖尋找一種能夠誘導(dǎo)均衡、有效的體液和細(xì)胞免疫反應(yīng)的免疫原。 本課題根據(jù)HIV-1 Env三維結(jié)構(gòu)中四種廣譜中和單抗的結(jié)合表位及前人經(jīng)驗(yàn)，設(shè)計四組突變，通過四組之間不同搭配設(shè)計五種突變組合：M2、M5-1、M5-2、M4和M7。在原始株及各突變組合的基礎(chǔ)上做V1V2區(qū)的刪除，設(shè)計另六種突變組合：dWt、dM2、dM5-1、dM5-2、dM4和dM7。通過環(huán)形誘變，DpnI篩選的方式得到這11種突變體，酶切初步鑒定，測序確定突變結(jié)果。提取原始及突變質(zhì)粒并轉(zhuǎn)染293T細(xì)胞，經(jīng)WesternBlot檢測體外表達(dá)情況。原始及突變質(zhì)粒免疫家兔，用五個不同廠家ELISA抗體檢測試劑盒檢測血清抗體水平，用假病毒單周期感染中和試驗(yàn)檢測其中和抗體水平，將ELISA抗體檢測結(jié)果與中和試驗(yàn)檢測結(jié)果做相關(guān)性分析。原始及突變質(zhì)粒免疫小鼠，取血分離血清，檢測中和抗體水平；取脾分離脾白細(xì)胞，以SHIVchn19肽庫和單肽Env34作為刺激物，用IFN—γELISPOT方法評價細(xì)胞免疫反應(yīng)。將原始及改造后env基因克隆入pcDNA~(TM)3.1 D／V5-His-TOPO(?)載體，WB檢測體外表達(dá)情況，與pSG3~(△Env)共轉(zhuǎn)染293T細(xì)胞，收集上清感染TZM-bl細(xì)胞，檢測改造對功能性假病毒形成的影響。 WB結(jié)果顯示D-GPEi的原始株及各突變克隆，以及克隆入pcDNA~(TM)3.1 D／V5-His-TOPO(?)載體的原始及突變env在體外都能正常表達(dá)，且表達(dá)量沒有明顯區(qū)別，突變株的Env條帶較原始株泳動速率提高，進(jìn)一步佐證了改造結(jié)果。家兔血清ELISA檢測結(jié)果表明，電穿孔介導(dǎo)DNA免疫家兔，與單純DNA免疫相比，其所用質(zhì)粒量少，免疫周期短，抗體水平高。不同廠家抗體檢測試劑盒檢測家兔四次免疫后血清中抗體水平，檢測的結(jié)果數(shù)值上雖然存在差異，但總體的趨勢相似。Wt、N2010、G2、M2、M5-1和dM5-1組抗體水平較高，但各組與Wt組比較沒有明顯優(yōu)勢。對于同一質(zhì)粒免疫的兩只家兔，其抗體水平不一致。中和試驗(yàn)結(jié)果顯示，Wt、N2010、G2、M2和dM2中和抗體水平較高，但同一組兩只家兔中和抗體水平不一致。將ELISA抗體檢測結(jié)果與假病毒中和試驗(yàn)檢測結(jié)果，做相關(guān)性分析，兩者之間沒有統(tǒng)計學(xué)意義上的相關(guān)性，但對于同一組兩只家兔，其中和抗體水平高的總抗體水平也高。小鼠ELISPOT結(jié)果顯示，與原始組相比，，改造組細(xì)胞免疫反應(yīng)大部分是降低的，包括M5-1、dM5-1、M5-2、dM5-2、M7、dM7、dWt、M4和dM4，其中與Wt組相比降低有統(tǒng)計學(xué)意義(p＜0.05)的包括dM5-1、M5-2、M7、dM7和dM4，其余各組與Wt組相比雖有降低但無統(tǒng)計學(xué)意義。改造組M2、dM2、G2與Wt組相比細(xì)胞免疫反應(yīng)增高，其中M2組的增高有統(tǒng)計學(xué)意義(p＜0.05)。針對四個肽庫的細(xì)胞免疫反應(yīng)由強(qiáng)到弱：肽庫1＞肽庫3＞肽庫2＞肽庫4，肽庫1在四肽庫總反應(yīng)中所占比例最高，＞75％。各肽庫反應(yīng)與總反應(yīng)的相關(guān)性由高到低肽庫1＞肽庫3＞肽庫2＞肽庫4，針對Env34的細(xì)胞免疫反應(yīng)與總反應(yīng)有較高的相關(guān)性，相關(guān)系數(shù)為0.942(p＜0.01)。在Wt和74-2兩假病毒中和試驗(yàn)中，dWt、M2、M5-2、M5-1、dM7、N201Q組與Wt組相比，其中和抗體水平有增高趨勢，只有dWt組和M5-2組的增高在兩假病毒試驗(yàn)中都有有統(tǒng)計學(xué)意義(p＜0.05)，此外，在Wt假病毒中和試驗(yàn)中，M5-1組的增高有統(tǒng)計學(xué)意義，在74-2假病毒中和試驗(yàn)中，M2和N2010組的增高有統(tǒng)計學(xué)意義。從兩假病毒中和試驗(yàn)結(jié)果可以看出，dM2、dM5-2、M4、dM4和M7組中和抗體水平與Wt組相比有降低趨勢，其中只有在Wt假病毒中和試驗(yàn)中，M4、dM4組的降低有統(tǒng)計學(xué)意義。單周期感染實(shí)驗(yàn)結(jié)果顯示，除G2改造外，其它改造都使功能性假病毒形成能力大為下降。 從動物實(shí)驗(yàn)結(jié)果可以得出：不同改造對Env免疫原性的影響不同，M2組改造使Env誘導(dǎo)細(xì)胞與體液免疫的能力都有一定的提高；dWt組改造，使誘導(dǎo)體液免疫的能力有一定幅度提高，同時誘導(dǎo)細(xì)胞免疫的能力沒有明顯變化；N2010、M5-1、M5-2和dM7雖然誘導(dǎo)細(xì)胞免疫能力有所下降，但誘導(dǎo)體液免疫能力有一定程度提高；dM2和G2誘導(dǎo)體液免疫能力降低，但誘導(dǎo)細(xì)胞免疫能力有小幅度提高或基本不變；dM5-2、M4、dM4和M7組改造使其誘導(dǎo)細(xì)胞及體液免疫的能力都降低。從單周期感染實(shí)驗(yàn)結(jié)果可以得出：G2(N620Q，N632Q)改造不影響功能性假病毒的形成，而F174A和N2010兩個單點(diǎn)的改造使Env喪失了形成功能性假病毒的能力。
[Abstract]:The membrane protein (Env) of the human immunodeficiency virus (HIV) is located on the surface of the virus, is the main target for neutralizing the neutralizing antibody, but the wild-type Env is susceptible to immune escape under the body's immune pressure, which may be associated with its complex conformation and the surface oligose chain. The present subject attempts to expose potential neutralizing antibody epitopes in the currently known and CD4i (CD4-induced) region by removing the oligosaccharide chain, the complex amino acid side chain and the high-transformation region, with a view to inducing a broad-spectrum and effective neutralizing antibody, while the humoral immunity is combined with cellular immunity, The invention seeks to find an immunogen capable of inducing a balanced, effective humoral and cellular immune response. According to the binding epitopes of four broad-spectrum neutralizing monoclonal antibodies in the three-dimensional structure of HIV-1 Env and the previous experience, four groups of mutations were designed, and five mutation combinations were designed by different collocations among the four groups: M2, M5-1, M5-2. on the basis of the original plant and each mutation combination, the deletion of the V1V2 region is carried out, and the other six mutation combinations are designed: dWt, dM2, dM5-1, dM5-2 and d M4 and dM7. The 11 mutants were obtained by ring-directed mutagenesis and DpnI screening, and the enzyme was initially identified and sequenced. The mutation results were determined. The original and mutant plasmids were extracted and the 293T cells were transfected and tested by Western Blot In vitro expression, the original and the mutant plasmid were immunized with rabbit, the serum antibody level was detected by using five different manufacturers of ELISA antibody detection kit, and the ELISA antibody detection result is compared with the neutralization test detection result by using the false virus single-cycle infection and the test to detect the antibody level. Carry out the correlation analysis. The original and mutant plasmid was used to immunize the mice, and the serum, the serum and the antibody level were isolated from the blood, and the spleen and the white blood cells were isolated from the spleen, and the SHIVchn19 peptide library and the single peptide Env34 were used as the stimulus, and the method was evaluated using the method of the method of the IFN-CREELISPOT. Cellular immune response. The original and modified env gene was cloned into pcDNA ~ (TM) 3.1D/ V5-His - In vitro expression of TOPO (?) vector and WB, 293T cells were co-transfected with pSG3 ~ (+ Env), and the supernatant was collected to infect TZM-bl cells. The effects of virus formation. WB results showed that the original strain of D-GPEi and the mutant clones were cloned and cloned into pcDNA ~ (TM) 3.1D / The original and mutant env of the V5-His-TOPO (?) vector can be expressed in vitro, and the expression amount is not obviously different, and the Env band of the mutant strain is higher than that of the original plant. The results of the modified rabbit serum ELISA showed that the electroporation-mediated DNA-immunized rabbit, compared with the pure DNA immunization, used the plasmid quantity. in that invention, the antibody level in the serum after four immunization of the rabbit is detected by the antibody detection kit of different manufacturers, and the result value of the detection is There was a difference, but the overall trend was similar. The levels of antibodies in Wt, N2010, G2, M2, M5-1, and dM5-1 groups were high, but each There was no significant advantage between the group and the Wt group. For the same plasmid, it was immune to the same plasmid. In both rabbits, the antibody levels were not consistent. The neutralization test showed that the levels of neutralizing antibodies in Wt, N2010, G2, M2, and dM2 were high, but the same group The level of the neutralizing antibodies in both rabbits was not consistent. The results of the detection of the ELISA antibody and the test results of the test and the correlation analysis were not statistically significant, but for the same group of two rabbits, and The high level of the total antibody was also high in the antibody level. The mouse ELISPOT results showed that the majority of the cell immune response in the modified group was reduced compared to the original group, including M5-1, dM5-1, M5-2, dM5-2, M7, dM7, dWt, M4, and dM4, with a statistically significant decrease compared to the Wt group (p <0.05), including dM5-1, M5. -2, M7, d7 and dM4, and the remaining groups and the Wt group In the modified group M2, dM2, and G2, the immune response of M2, dM2, and G2 was higher than that of the Wt group. Statistical significance (p <0.05). The cellular immune response to the four peptide libraries is from strong to weak: peptide library 1> peptide library 3> peptide library 2> peptide library 4, peptide library 1 in total anti-peptide library The correlation between the reaction of each peptide library and the total reaction is from high to low peptide library 1> peptide library 3> peptide library 2> peptide library 4, and the cellular immune response to Env34 has a high correlation with the total reaction, and the correlation coefficient is The levels of dWt, M2, M5-2, M5-1, d7 and N201Q were higher than that of the Wt group. Significance (p <0.05), and in addition, in the Wt pseudovirus and in the test, the increase in the M5-1 group was statistically significant, in the 74-2 pseudovirus and in the test, M2 and N2 The elevation of the 010 group was statistically significant. It can be seen from both the two pseudoviruses and the test results that the levels of dM2, dM5-2, M4, dM4, and M7 have a tendency to decrease as compared to the Wt group, where only in the Wt pseudovirus and in the test, M4, The decrease of dM4 group is of statistical significance. The results of single-cycle infection show that, in addition to the transformation of G2, other modifications make work The results of animal experiments show that the effects of different modifications on the immunogenicity of Env are different, and the transformation of M2 group has a certain increase in the ability of Env to induce the immunity of Env, and the dWt group The ability of inducing humoral immunity is improved, and the ability of inducing cell immunity is not changed. N2010, M5-1, M5-2 and d7 have a certain effect on the immune competence of the cells. Increased degree of immunity; dM2 and G2 induce a decrease in humoral immunity, but the induction of cellular immunity has a small increase or substantially the same; dM5-2, M4, dM4, and M7 The ability of group modification to induce cell and humoral immunity decreased. The results of single-cycle infection showed that the transformation of G2 (N620Q, N632Q) did not affect the formation of functional pseudovirus, while F174A and N2010 were both single-point.
【學(xué)位授予單位】：中國藥品生物制品檢定所
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392
本文編號：2505363
[Abstract]:The membrane protein (Env) of the human immunodeficiency virus (HIV) is located on the surface of the virus, is the main target for neutralizing the neutralizing antibody, but the wild-type Env is susceptible to immune escape under the body's immune pressure, which may be associated with its complex conformation and the surface oligose chain. The present subject attempts to expose potential neutralizing antibody epitopes in the currently known and CD4i (CD4-induced) region by removing the oligosaccharide chain, the complex amino acid side chain and the high-transformation region, with a view to inducing a broad-spectrum and effective neutralizing antibody, while the humoral immunity is combined with cellular immunity, The invention seeks to find an immunogen capable of inducing a balanced, effective humoral and cellular immune response. According to the binding epitopes of four broad-spectrum neutralizing monoclonal antibodies in the three-dimensional structure of HIV-1 Env and the previous experience, four groups of mutations were designed, and five mutation combinations were designed by different collocations among the four groups: M2, M5-1, M5-2. on the basis of the original plant and each mutation combination, the deletion of the V1V2 region is carried out, and the other six mutation combinations are designed: dWt, dM2, dM5-1, dM5-2 and d M4 and dM7. The 11 mutants were obtained by ring-directed mutagenesis and DpnI screening, and the enzyme was initially identified and sequenced. The mutation results were determined. The original and mutant plasmids were extracted and the 293T cells were transfected and tested by Western Blot In vitro expression, the original and the mutant plasmid were immunized with rabbit, the serum antibody level was detected by using five different manufacturers of ELISA antibody detection kit, and the ELISA antibody detection result is compared with the neutralization test detection result by using the false virus single-cycle infection and the test to detect the antibody level. Carry out the correlation analysis. The original and mutant plasmid was used to immunize the mice, and the serum, the serum and the antibody level were isolated from the blood, and the spleen and the white blood cells were isolated from the spleen, and the SHIVchn19 peptide library and the single peptide Env34 were used as the stimulus, and the method was evaluated using the method of the method of the IFN-CREELISPOT. Cellular immune response. The original and modified env gene was cloned into pcDNA ~ (TM) 3.1D/ V5-His - In vitro expression of TOPO (?) vector and WB, 293T cells were co-transfected with pSG3 ~ (+ Env), and the supernatant was collected to infect TZM-bl cells. The effects of virus formation. WB results showed that the original strain of D-GPEi and the mutant clones were cloned and cloned into pcDNA ~ (TM) 3.1D / The original and mutant env of the V5-His-TOPO (?) vector can be expressed in vitro, and the expression amount is not obviously different, and the Env band of the mutant strain is higher than that of the original plant. The results of the modified rabbit serum ELISA showed that the electroporation-mediated DNA-immunized rabbit, compared with the pure DNA immunization, used the plasmid quantity. in that invention, the antibody level in the serum after four immunization of the rabbit is detected by the antibody detection kit of different manufacturers, and the result value of the detection is There was a difference, but the overall trend was similar. The levels of antibodies in Wt, N2010, G2, M2, M5-1, and dM5-1 groups were high, but each There was no significant advantage between the group and the Wt group. For the same plasmid, it was immune to the same plasmid. In both rabbits, the antibody levels were not consistent. The neutralization test showed that the levels of neutralizing antibodies in Wt, N2010, G2, M2, and dM2 were high, but the same group The level of the neutralizing antibodies in both rabbits was not consistent. The results of the detection of the ELISA antibody and the test results of the test and the correlation analysis were not statistically significant, but for the same group of two rabbits, and The high level of the total antibody was also high in the antibody level. The mouse ELISPOT results showed that the majority of the cell immune response in the modified group was reduced compared to the original group, including M5-1, dM5-1, M5-2, dM5-2, M7, dM7, dWt, M4, and dM4, with a statistically significant decrease compared to the Wt group (p <0.05), including dM5-1, M5. -2, M7, d7 and dM4, and the remaining groups and the Wt group In the modified group M2, dM2, and G2, the immune response of M2, dM2, and G2 was higher than that of the Wt group. Statistical significance (p <0.05). The cellular immune response to the four peptide libraries is from strong to weak: peptide library 1> peptide library 3> peptide library 2> peptide library 4, peptide library 1 in total anti-peptide library The correlation between the reaction of each peptide library and the total reaction is from high to low peptide library 1> peptide library 3> peptide library 2> peptide library 4, and the cellular immune response to Env34 has a high correlation with the total reaction, and the correlation coefficient is The levels of dWt, M2, M5-2, M5-1, d7 and N201Q were higher than that of the Wt group. Significance (p <0.05), and in addition, in the Wt pseudovirus and in the test, the increase in the M5-1 group was statistically significant, in the 74-2 pseudovirus and in the test, M2 and N2 The elevation of the 010 group was statistically significant. It can be seen from both the two pseudoviruses and the test results that the levels of dM2, dM5-2, M4, dM4, and M7 have a tendency to decrease as compared to the Wt group, where only in the Wt pseudovirus and in the test, M4, The decrease of dM4 group is of statistical significance. The results of single-cycle infection show that, in addition to the transformation of G2, other modifications make work The results of animal experiments show that the effects of different modifications on the immunogenicity of Env are different, and the transformation of M2 group has a certain increase in the ability of Env to induce the immunity of Env, and the dWt group The ability of inducing humoral immunity is improved, and the ability of inducing cell immunity is not changed. N2010, M5-1, M5-2 and d7 have a certain effect on the immune competence of the cells. Increased degree of immunity; dM2 and G2 induce a decrease in humoral immunity, but the induction of cellular immunity has a small increase or substantially the same; dM5-2, M4, dM4, and M7 The ability of group modification to induce cell and humoral immunity decreased. The results of single-cycle infection showed that the transformation of G2 (N620Q, N632Q) did not affect the formation of functional pseudovirus, while F174A and N2010 were both single-point.
【學(xué)位授予單位】：中國藥品生物制品檢定所
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392
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