胃癌MG7-Ag模拟表位为基础构建多表位联合的基因疫苗的研究
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摘要
胃癌是严重威胁我国人民健康的恶性肿瘤，目前胃癌的治疗手段主要有手术切除和化疗，但均不能有效解决肿瘤转移、复发等问题。免疫治疗是肿瘤治疗的新方法，包括用外源性抗体治疗、用细胞因子调节免疫以及主动免疫治疗，用疫苗对患者的免疫系统产生特异性刺激。目前尚无有效的疫苗用于胃癌的主动免疫治疗。胃癌MG7抗原是本研究所在八十年代发现的特异性较高的肿瘤标志物，抗原分析表明其抗原决定簇位于糖链上。由于糖抗原免疫原性弱，纯化、制备困难，且不易诱导T细胞免疫反应。用分子模拟多肽替代糖抗原进行免疫治疗是解决问题的方法之一，并在动物模型中取得很好的效果。本研究所用噬菌体随机肽库技术筛选得到了MG7抗原的模拟肽表位，抗体竞争结合抑制实验证明模拟肽表位可以有效地模拟天然抗原，计算机辅助分析显示模拟表位可与HLA分子结合，这为胃癌疫苗的研究奠定了基础。
DNA疫苗具有诱导特异、高效、持久的免疫反应，制备简便
第四军医大学硕士学位论文
等特点，已广泛用于肿瘤疫苗的基础研究。特别是可以模拟内源性
抗原的加工呈递过程，诱发特异性细胞免疫反应。其免疫途径有肌
肉注射、皮下注射、静脉注射等。目前进入临床实验的质粒DNA
疫苗有针对黑色素瘤、B细胞淋巴瘤、人类免疫缺陷病毒、乙肝病
毒和疟原虫等疾病，多使用肌肉注射。安全性实验表明，人体对
DNA疫苗耐受良好。
为了增强以表位为基础的DNA疫苗的免疫效果，常采用的策
略有:辅助因子的融合或共注射:抗原表位基因与载体蛋白融合;
多表位联合组建疫苗等。
多个表位联合的疫苗可以诱导机体产生针对各个不同表位的
抗原特异性免疫反应，重复单一表位可通过增加表位的拷贝数和加
强Thl细胞反应而提高疫苗的免疫效能。在设计多表位串联疫苗
时，表位的侧翼序列对于表位的免疫原性影响很大，故在表位之间
添加间隔序列(sPacer)非常关键。合适的sPacer能避免表位间的
相互干扰，以及联合表位与MHC分子的非特异性结合;对于蛋白
酶对抗原肤水解也有影响。
抗原模拟表位多为8一12多肤，在体内容易被降解，故难以激
发有效的免疫反应，因此应选择适当的载体或佐剂保护抗原表位，
并促进其递呈给T淋巴细胞，诱导特异性的免疫反应。热休克蛋
白70(HSP7O)是MHCI类分子抗原呈递途径中的一种伴侣分子，
在未与MHCI类分子结合之前，经蛋白酶体切割的内源性肤段很
不稳定，而HSP70可与肤段结合，稳定肤段并将其送到内质网与
TAP分子结合。HSP能够将肿瘤抗原肤转运至宿主的APC，再呈
第四军医大学硕士学位论文
递给T细胞，从而促进抗原特异性免疫反应。HSP本身具有免疫
刺激剂的作用，可增加多种细胞因子的分泌。HSP70在各种生物体
中高度保守，不易引起异体间的免疫反应，不会干扰抗原特异性免
疫的结果。因此，HSP70被认为是一种良好的免疫佐剂，可用于
肿瘤疫苗的研究。以HSP7O为基础的疫苗已进入临床试验阶段。
从肿瘤中提纯的HSP7O一肤复合物能引起机体产生特异性抗瘤反
应。体外重组的HSP70一肤复合物或HSP7O与抗原表位的融合基因
均能诱导抗原特异性的免疫反应，并进行免疫治疗。
趋化因子作为肿瘤疫苗的佐剂是增强疫苗效能的另一途径，在
感染性疾病的疫苗研究中已有用。趋化因子MIG和IP一10不仅能
够募集T细胞、NK细胞等，或将抗原肤段靶向转染APC细胞，
还具有抑制血管生成的作用，可从双方面提高治疗肿瘤的效果。但
是，趋化因子在肿瘤生物学中具有广泛的功能，可能通过与肿瘤细
胞表达的趋化因子受体作用，在肿瘤的白细胞浸润的表型、新生血
管生成、肿瘤细胞的生长、存活和转移等方面其重要作用。因此，
在应用MIG或IP一10治疗胃癌之前，应首先明确胃癌中其受体
CXC3的表达情况，以预测应用后的作用机理和效果。
目的以HSP7O为载体，GGGS为间隔序列，构建含有4个
串联的MG7一Ag模拟表位与HSP70的融合基因疫苗，并观察该疫
苗能否诱导BALB/c小鼠产生特异性体液免疫、细胞免疫及其体内
抗瘤效应。检测胃癌组织中趋化因子受体CXCR3的表达水平。
方法1.将4个不同的胃癌MG7一Ag模拟表位依次设计于下
游引物中，通过多次PCR将表位串联于一段无关的载体基因，将
第四军医大学硕士学位论文
目的基因插入PMD一1 ST载体，酶切鉴定，测序后，利用BamHI
和EeoRI将基因亚克隆入真核表达载体peDNA3 .IB载体，Hind
111酶切除无关载体基因，用EcoRI和Xhol将热休克蛋白基因插
入表位之后作为佐剂，得到多表位串联的DNA疫苗。采用脂质体
转染的方法将重组质粒瞬时转染至小鼠骨髓瘤SPZ/0细胞。W已stem
blotting鉴定重组蛋白4MG7一HSP70的表达2.
peDNA3 .IB/4MG7一HSP70基因疫苗采用肌肉注射和静脉注射两种
方式免疫接种BALB/c小鼠，每隔2周加强免疫一次，共免疫3次。
以生理盐水、空载体质粒、peDNA3 .1(+)/HSP70质粒和
pcDNA3.l(+)/IMG一HSP70质粒为对照。分别于初次免疫后2、4、
6周收集血清，进行ELISA。第6周时取小鼠脾细胞，用MTT法
进行体外杀伤实验，检测?
Background Gastric cancer is among the most common malignant tumors in China and is mainly treated by surgery and chemotherapy. However, those conventional therapies cannot deal with such problems as tumor metastasis and recurrence. Cancer immunotherapy is a rather new field of tumor therapy, which includes monoclonal antibodies, tumor vaccines, immune response modifiers, such as Interferon-a, Interleukin-2 and Interleukin-12. But now there is no effective gastric cancer vaccine developed yet. MG7-Ag is a specific marker molecule of gastric cancer, which was discovered by our institute. Its antigenicity was found to locate in a sugar chain, which means it is a carbohydrate antigen. Carbohydrate antigens are intrinsically T- cell-independent antigen, often eliciting a short-lived IgM response of poor memory without inducing a T-cell response. It is also hard to purify or mass-produce carbohydrate antigen. One way to
solve these problems is to substitute carbohydrate antigen with peptides that could mimic the original antigen, which has been proved in many animal experiments. By screening the phage display library, a series of mimicry peptides were identified in our institute. Competitive antibody binding assay indicated that the mimicry peptides could mimic MG7-Ag well, and analysis with computer showed that the mimicry peptides could bind with HLA molecules.
DNA vaccine is now widely used in lab research because of its ability to induce specific, effective and persisting immunity. It also can mimic the processing and presenting of endogenous antigen, thus inducing T cell response. The immunizing pathways of DNA vaccine include intramuscular injection, intradermal injection, skin gene gun bombard and intraveinous injection. Many DNA vaccines have been evaluated in clinical trials, such as DNA vaccines against melanoma, B cell lymphoma, HIV, HBV and plasmodium, in which most of them were given by intramuscular injection. It is implicated that the DNA vaccine are well tolerated by human body.
To enhance the effect of epitope-based gene immunization, several strategies could be adopted: fusion or coinjection of cytokines; fusion of the epitope to a carrier protein; combination of multiple epitopes, etc.
Multi-epitope vaccine induces specific immune response against each epitope in the vaccine. And the potency of repeated-epitope
vaccine could be improved because the epitope's copy is increased and Thl response is enhanced. When several epitopes are combined together, flanking sequence will affect the potency of the vaccine. Therefore, it is crucial to insert spacers between epitopes. Proper spacer can prevent mutual interference between epitopes and unspecific binding with MHC molecules.
Oligopeptides are degraded easily in vivo and require carrier proteins to protect them. Heat shock protein 70 is a molecular chaperon in the pathway of MHC I-dependent antigen processing. It can stabilize the peptide and transfer it to endoplasm reticulum. Acting as a immune stimulator, HSP70 can up regulate the expression level of cytokines. Therefore, HSP70 is a good carrier of vaccine. Vaccines based on HSP70 have been explored in clinical research. The HSP70-peptide complex derived from tumor tissues or recombined in vitro can induce specific anti-tumor response. Similar effect could be obtained with the fusion gene of HSP70 and peptides.
Another approach to improve the immunogenicity of DNA vaccine is through the co-delivery of chemokine expression plasmid or fusion of the antigen to chemokines. Studies in a variety of infectioius disease clearly demonstrate that chemokine could alter the magnititude and direction of the immune response elicited by DNA vaccine. MIG and IP-10 not only recruit immune cells such as T cells, NK cells, but also inhibit angiogenesis, which makes them especially fit for tumor
vaccine. However, many human involve a complex chemokine network that may influence the extent and phenotype of the leukocyte infiltration, angiogenesis, tumor cell growth , survival and metastasis. Chemokine rereptors exp
DNA疫苗具有诱导特异、高效、持久的免疫反应，制备简便
第四军医大学硕士学位论文
等特点，已广泛用于肿瘤疫苗的基础研究。特别是可以模拟内源性
抗原的加工呈递过程，诱发特异性细胞免疫反应。其免疫途径有肌
肉注射、皮下注射、静脉注射等。目前进入临床实验的质粒DNA
疫苗有针对黑色素瘤、B细胞淋巴瘤、人类免疫缺陷病毒、乙肝病
毒和疟原虫等疾病，多使用肌肉注射。安全性实验表明，人体对
DNA疫苗耐受良好。
为了增强以表位为基础的DNA疫苗的免疫效果，常采用的策
略有:辅助因子的融合或共注射:抗原表位基因与载体蛋白融合;
多表位联合组建疫苗等。
多个表位联合的疫苗可以诱导机体产生针对各个不同表位的
抗原特异性免疫反应，重复单一表位可通过增加表位的拷贝数和加
强Thl细胞反应而提高疫苗的免疫效能。在设计多表位串联疫苗
时，表位的侧翼序列对于表位的免疫原性影响很大，故在表位之间
添加间隔序列(sPacer)非常关键。合适的sPacer能避免表位间的
相互干扰，以及联合表位与MHC分子的非特异性结合;对于蛋白
酶对抗原肤水解也有影响。
抗原模拟表位多为8一12多肤，在体内容易被降解，故难以激
发有效的免疫反应，因此应选择适当的载体或佐剂保护抗原表位，
并促进其递呈给T淋巴细胞，诱导特异性的免疫反应。热休克蛋
白70(HSP7O)是MHCI类分子抗原呈递途径中的一种伴侣分子，
在未与MHCI类分子结合之前，经蛋白酶体切割的内源性肤段很
不稳定，而HSP70可与肤段结合，稳定肤段并将其送到内质网与
TAP分子结合。HSP能够将肿瘤抗原肤转运至宿主的APC，再呈
第四军医大学硕士学位论文
递给T细胞，从而促进抗原特异性免疫反应。HSP本身具有免疫
刺激剂的作用，可增加多种细胞因子的分泌。HSP70在各种生物体
中高度保守，不易引起异体间的免疫反应，不会干扰抗原特异性免
疫的结果。因此，HSP70被认为是一种良好的免疫佐剂，可用于
肿瘤疫苗的研究。以HSP7O为基础的疫苗已进入临床试验阶段。
从肿瘤中提纯的HSP7O一肤复合物能引起机体产生特异性抗瘤反
应。体外重组的HSP70一肤复合物或HSP7O与抗原表位的融合基因
均能诱导抗原特异性的免疫反应，并进行免疫治疗。
趋化因子作为肿瘤疫苗的佐剂是增强疫苗效能的另一途径，在
感染性疾病的疫苗研究中已有用。趋化因子MIG和IP一10不仅能
够募集T细胞、NK细胞等，或将抗原肤段靶向转染APC细胞，
还具有抑制血管生成的作用，可从双方面提高治疗肿瘤的效果。但
是，趋化因子在肿瘤生物学中具有广泛的功能，可能通过与肿瘤细
胞表达的趋化因子受体作用，在肿瘤的白细胞浸润的表型、新生血
管生成、肿瘤细胞的生长、存活和转移等方面其重要作用。因此，
在应用MIG或IP一10治疗胃癌之前，应首先明确胃癌中其受体
CXC3的表达情况，以预测应用后的作用机理和效果。
目的以HSP7O为载体，GGGS为间隔序列，构建含有4个
串联的MG7一Ag模拟表位与HSP70的融合基因疫苗，并观察该疫
苗能否诱导BALB/c小鼠产生特异性体液免疫、细胞免疫及其体内
抗瘤效应。检测胃癌组织中趋化因子受体CXCR3的表达水平。
方法1.将4个不同的胃癌MG7一Ag模拟表位依次设计于下
游引物中，通过多次PCR将表位串联于一段无关的载体基因，将
第四军医大学硕士学位论文
目的基因插入PMD一1 ST载体，酶切鉴定，测序后，利用BamHI
和EeoRI将基因亚克隆入真核表达载体peDNA3 .IB载体，Hind
111酶切除无关载体基因，用EcoRI和Xhol将热休克蛋白基因插
入表位之后作为佐剂，得到多表位串联的DNA疫苗。采用脂质体
转染的方法将重组质粒瞬时转染至小鼠骨髓瘤SPZ/0细胞。W已stem
blotting鉴定重组蛋白4MG7一HSP70的表达2.
peDNA3 .IB/4MG7一HSP70基因疫苗采用肌肉注射和静脉注射两种
方式免疫接种BALB/c小鼠，每隔2周加强免疫一次，共免疫3次。
以生理盐水、空载体质粒、peDNA3 .1(+)/HSP70质粒和
pcDNA3.l(+)/IMG一HSP70质粒为对照。分别于初次免疫后2、4、
6周收集血清，进行ELISA。第6周时取小鼠脾细胞，用MTT法
进行体外杀伤实验，检测?
Background Gastric cancer is among the most common malignant tumors in China and is mainly treated by surgery and chemotherapy. However, those conventional therapies cannot deal with such problems as tumor metastasis and recurrence. Cancer immunotherapy is a rather new field of tumor therapy, which includes monoclonal antibodies, tumor vaccines, immune response modifiers, such as Interferon-a, Interleukin-2 and Interleukin-12. But now there is no effective gastric cancer vaccine developed yet. MG7-Ag is a specific marker molecule of gastric cancer, which was discovered by our institute. Its antigenicity was found to locate in a sugar chain, which means it is a carbohydrate antigen. Carbohydrate antigens are intrinsically T- cell-independent antigen, often eliciting a short-lived IgM response of poor memory without inducing a T-cell response. It is also hard to purify or mass-produce carbohydrate antigen. One way to
solve these problems is to substitute carbohydrate antigen with peptides that could mimic the original antigen, which has been proved in many animal experiments. By screening the phage display library, a series of mimicry peptides were identified in our institute. Competitive antibody binding assay indicated that the mimicry peptides could mimic MG7-Ag well, and analysis with computer showed that the mimicry peptides could bind with HLA molecules.
DNA vaccine is now widely used in lab research because of its ability to induce specific, effective and persisting immunity. It also can mimic the processing and presenting of endogenous antigen, thus inducing T cell response. The immunizing pathways of DNA vaccine include intramuscular injection, intradermal injection, skin gene gun bombard and intraveinous injection. Many DNA vaccines have been evaluated in clinical trials, such as DNA vaccines against melanoma, B cell lymphoma, HIV, HBV and plasmodium, in which most of them were given by intramuscular injection. It is implicated that the DNA vaccine are well tolerated by human body.
To enhance the effect of epitope-based gene immunization, several strategies could be adopted: fusion or coinjection of cytokines; fusion of the epitope to a carrier protein; combination of multiple epitopes, etc.
Multi-epitope vaccine induces specific immune response against each epitope in the vaccine. And the potency of repeated-epitope
vaccine could be improved because the epitope's copy is increased and Thl response is enhanced. When several epitopes are combined together, flanking sequence will affect the potency of the vaccine. Therefore, it is crucial to insert spacers between epitopes. Proper spacer can prevent mutual interference between epitopes and unspecific binding with MHC molecules.
Oligopeptides are degraded easily in vivo and require carrier proteins to protect them. Heat shock protein 70 is a molecular chaperon in the pathway of MHC I-dependent antigen processing. It can stabilize the peptide and transfer it to endoplasm reticulum. Acting as a immune stimulator, HSP70 can up regulate the expression level of cytokines. Therefore, HSP70 is a good carrier of vaccine. Vaccines based on HSP70 have been explored in clinical research. The HSP70-peptide complex derived from tumor tissues or recombined in vitro can induce specific anti-tumor response. Similar effect could be obtained with the fusion gene of HSP70 and peptides.
Another approach to improve the immunogenicity of DNA vaccine is through the co-delivery of chemokine expression plasmid or fusion of the antigen to chemokines. Studies in a variety of infectioius disease clearly demonstrate that chemokine could alter the magnititude and direction of the immune response elicited by DNA vaccine. MIG and IP-10 not only recruit immune cells such as T cells, NK cells, but also inhibit angiogenesis, which makes them especially fit for tumor
vaccine. However, many human involve a complex chemokine network that may influence the extent and phenotype of the leukocyte infiltration, angiogenesis, tumor cell growth , survival and metastasis. Chemokine rereptors exp
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