旋毛虫与Sp2/0骨髓瘤细胞相关抗原基因抗肿瘤效应研究
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摘要
为筛选旋毛虫与Sp2/0骨髓瘤细胞相关抗原基因,观察其抗肿瘤效果,以探讨其抗肿瘤机制,本研究首先构建了旋毛虫cDNA表达文库,用抗Sp2/0骨髓瘤细胞的多克隆抗体对文库进行筛选,获得旋毛虫与Sp2/0骨髓瘤细胞相关抗原基因,利用5’-RACE技术进行末端快速扩增获得全长后,将该基因在真核表达载体中表达。应用斑点杂交技术对相关抗原基因进行定位,并进行小鼠抗肿瘤试验。实验结果表明,成功构建了旋毛虫肌幼虫cDNA表达文库,文库容量为1.9×106pfu/mL,重组率为97.5%,扩增后文库滴度为1.6×109pfu/mL。经过3轮筛选,获得一个旋毛虫肌幼虫相关抗原基因片段,通过5’-RACE扩增该基因全长,测序结果显示TS2全长为569bp,含有一大小为411bp的开放阅读框。将其命名为TS2。登陆NCBI进行Blast比对发现该基因为核糖体蛋白S24基因,与DNA修复、细胞发育调控和细胞分化有关。ELM服务器分析结构域,表明此序列蛋白质含有4个精氨酸二元转移酶切割位点,1个羧基肽酰胺化位点和1个氨基葡聚糖附着位点。核酸疫苗重组质粒pVAX1-TS2转染Hela细胞,PCR和Western blotting检测结果表明目的基因在其中得到正确转录和表达,表达蛋白具有反应原性。应用核酸疫苗重组质粒免疫小鼠进行的体内抗肿瘤试验表明,试验组小鼠产生了特异的细胞免疫应答,CD3+、CD4+T淋巴细胞均进行了有效增殖。试验组和对照组肿瘤体积和重量的统计学分析比较表明,旋毛虫核酸疫苗重组质粒的抑瘤效果明显高于对照组。
Tumor has become the primary threat in modern human life. The statistical data in 2000 from WHO indicated that there were 10 060 000 people infected by cancers globally and 6200 000 people died. In China, the number of cancer patients is increasing at the rate of 3 percent per year based on 2 million. Prevention and cure on cancer have become a very important task. With the development of biotechnology and thorough study in molecular mechanism of tumor genesis, biotherapy has become the fourth mode after radiotherapy,chemotherapy,operative treatment to cure cancer. It’s very important to improve the therapeutic effect and life quality by combining biotherapy and other therapeutic techniques, which is been facial to biotherapy in the combined therapy to tumor. International studies have demonstrated that there were anti-tumor active components in many marine animals and plants. Lots of viruses and parasites also have anti-tumor effects. Until recently, some studies have shown that a lot of parasites have anti-tumor effect including Trichinella spiralis, acantho-amoeba trophozoite, plasmodium et al but the mechanism involved is unknown.
Trichinosis is a globalized zoontic parasitic disease. Trichinella spiralis can infect the human and 150 kinds of animals including birds and rare animals. Some previous study discovered that the immunity of hosts was enhanced after infected by Trichinella spiralis. That Trichinella spiralis can activate the host’s immune system is an important factor, meanwhile it probably contain some antitumor components. In the present study we have constructed cDNA express library of Trichella spiralis and identified a novel related protein gene by screening this library with anti-serum of Sp2/0 Myeloma Cell. This gene was designated TS2. The responses of specific cell immunity in mice were elevated by muscle injecting TS2 recombined plasmid incubating Sp2/0 Myeloma Cell in mice. The results suggested that the TS2 gene may be an important antigen gene possessing antitumor biological activity. This study has established foundations for developing some new antitumor vaccine.
Construction of cDNA express library on Trichinella spiralis The cDNA library was a basis for researching structure, expression and function of genes. In this study, in order to study antitumor function of related gene between Trichinella spiralis and Sp2/0 Myeloma cell, cDNA express Library of Trichinella spiralis muscle larvae was constructed. Total RNA and mRNA of Trichinella spiralis were isolated and purified. cDNAs were synthesized by reverse transcription. Fragments shorter than 400bp were removed by low melting-point agarose gel after adding EcoR I adaptors and Phosphorylating and Digesting with Xho I. After ligation of the cDNA withλ-ZAP vector, recombinant lambda phage was packaged using Gigapack III Gold packaging extract. The data showed that approximately 97.5% of the library was recombinant. The titer of the amplied library was 1.9×106 pfu/mL. and insert DNA fragments were between 0.5-2.0kb by PCR with phage of randomly clones.
The preparation of polyclone’s antibody of Sp2/0 Myeloma cell and immunoscreening of related antigen gene Collecting Sp2/0 Myeloma cell of logarithmic growth phase and hyperacoustic quassation after freeze thawing 5 times repeatedly to prepare crude antigen. The polyclonal antibody for Trichinella spiralis anti Sp2/0 Myeloma cell was prepared through immunized inbred line Balb/C mouse by above antigen. The titers of serum tested by indirect ELISA are 1:8000. The serum can finely specifically bind with Trichinella spiralis muscle larvae by Western-blotting.
After the library was diluted and mixed with 300μl XL1-blue bacteria of OD600=1.0,inpouring mixture into NZY (Tet)preheated 45℃. the plate was cultured for 4 hours at 37℃at least. When the negative colony in the plate was grown up to needlepoint size in diameter, stick the nitrocellulose filters (NC) treated with IPTG to negative colony and culture for 8-10 hours at 37℃for transferred negative colony on NC. After the NC were washed and closed, then put into the above positive serum. After adding goat anti mouse enzyme labeling second antibody IgG and DAB coloration, one positive clone was obtained after three rounds immunoscreening. The fragment was amplified using T3 and T7 primers and phage as templates. Recombinant phage was excised in vivo using the ExAssist helper phage with SOLR strain. The sequencing results showed that this insert was 393 bp in length and contained a ploy (A) tail. In order to obtain total length included ORF, the fragment was amplified by 5'-RACE. The cDNA fragment was cloned into pMD-T18 vector and sequenced. The sequencing results showed that this insert was 569bp in length and contained a single open reading frame (ORF) and it was predicted to encode 136 amino acids. This gene was designated TS2 and was gene of Trichinella spiralis by BLAST searches with the GenBank database.ELM analysis showed that TS2 protein has four N-Arg dibasic convertase (nardilysine) cleavage site,one peptide C-terminal amidation site,one glycosaminoglycan attachment site,DNAStar software analysis in antigenic index showed that TS2 protein has six epitope sites. dot blot hybridization demonstrated that TS2 gene was not from myeloma cell but Trichinella spiralis.
Eukaryotic expression of the TS2 gene The fragment of TS2 ORF was amplified by PCR with designed primers according to TS2. The recombinant plasmids pMD18-T-TS2 was constructed. The ligation products were transformed to competent cells of host strain DH5α. The recombinant plasmids pVAX1-TS2 was constructed and transformed into E.coli Rosetta(DH5α)The recombiant plasmids pVAX1-TS2 was expressed in Hela cell strain. After transfected it into Hela cells, the specific recombinant proteins were detected in supernatant of Hela cell by Western blotting.
Anti-tumor effect of Trichinella spiralis on Sp2/0 myeloma cell in BalB/C mice The antitumor effect of TS2 protein against Sp2/0 myeloma cell was studied after the mice were inoculated with Sp2/0 myeloma cell after the mice intramuscular injected with recombinant plasmid, The results showed that recombinant plasmid couldn’t induce response of cellular immunity in mice. The mice inoculated indicated that the group vaccinated with pVAX1-TS2 elicited CD3+、CD4+ T subtype cells significant higher than that of the groups vaccinated with pVAX1 and PBS. The tumor size and the tumor weight of the group injected with recombinant plasmid of the TS2 were notable smaller than that of the groups injected with control plasmid and PBS. The above results suggested that the TS2 protein have better antitumor effect on Sp2/0 myeloma in mice.
Tumor has become the primary threat in modern human life. The statistical data in 2000 from WHO indicated that there were 10 060 000 people infected by cancers globally and 6200 000 people died. In China, the number of cancer patients is increasing at the rate of 3 percent per year based on 2 million. Prevention and cure on cancer have become a very important task. With the development of biotechnology and thorough study in molecular mechanism of tumor genesis, biotherapy has become the fourth mode after radiotherapy,chemotherapy,operative treatment to cure cancer. It’s very important to improve the therapeutic effect and life quality by combining biotherapy and other therapeutic techniques, which is been facial to biotherapy in the combined therapy to tumor. International studies have demonstrated that there were anti-tumor active components in many marine animals and plants. Lots of viruses and parasites also have anti-tumor effects. Until recently, some studies have shown that a lot of parasites have anti-tumor effect including Trichinella spiralis, acantho-amoeba trophozoite, plasmodium et al but the mechanism involved is unknown.
Trichinosis is a globalized zoontic parasitic disease. Trichinella spiralis can infect the human and 150 kinds of animals including birds and rare animals. Some previous study discovered that the immunity of hosts was enhanced after infected by Trichinella spiralis. That Trichinella spiralis can activate the host’s immune system is an important factor, meanwhile it probably contain some antitumor components. In the present study we have constructed cDNA express library of Trichella spiralis and identified a novel related protein gene by screening this library with anti-serum of Sp2/0 Myeloma Cell. This gene was designated TS2. The responses of specific cell immunity in mice were elevated by muscle injecting TS2 recombined plasmid incubating Sp2/0 Myeloma Cell in mice. The results suggested that the TS2 gene may be an important antigen gene possessing antitumor biological activity. This study has established foundations for developing some new antitumor vaccine.
Construction of cDNA express library on Trichinella spiralis The cDNA library was a basis for researching structure, expression and function of genes. In this study, in order to study antitumor function of related gene between Trichinella spiralis and Sp2/0 Myeloma cell, cDNA express Library of Trichinella spiralis muscle larvae was constructed. Total RNA and mRNA of Trichinella spiralis were isolated and purified. cDNAs were synthesized by reverse transcription. Fragments shorter than 400bp were removed by low melting-point agarose gel after adding EcoR I adaptors and Phosphorylating and Digesting with Xho I. After ligation of the cDNA withλ-ZAP vector, recombinant lambda phage was packaged using Gigapack III Gold packaging extract. The data showed that approximately 97.5% of the library was recombinant. The titer of the amplied library was 1.9×106 pfu/mL. and insert DNA fragments were between 0.5-2.0kb by PCR with phage of randomly clones.
The preparation of polyclone’s antibody of Sp2/0 Myeloma cell and immunoscreening of related antigen gene Collecting Sp2/0 Myeloma cell of logarithmic growth phase and hyperacoustic quassation after freeze thawing 5 times repeatedly to prepare crude antigen. The polyclonal antibody for Trichinella spiralis anti Sp2/0 Myeloma cell was prepared through immunized inbred line Balb/C mouse by above antigen. The titers of serum tested by indirect ELISA are 1:8000. The serum can finely specifically bind with Trichinella spiralis muscle larvae by Western-blotting.
After the library was diluted and mixed with 300μl XL1-blue bacteria of OD600=1.0,inpouring mixture into NZY (Tet)preheated 45℃. the plate was cultured for 4 hours at 37℃at least. When the negative colony in the plate was grown up to needlepoint size in diameter, stick the nitrocellulose filters (NC) treated with IPTG to negative colony and culture for 8-10 hours at 37℃for transferred negative colony on NC. After the NC were washed and closed, then put into the above positive serum. After adding goat anti mouse enzyme labeling second antibody IgG and DAB coloration, one positive clone was obtained after three rounds immunoscreening. The fragment was amplified using T3 and T7 primers and phage as templates. Recombinant phage was excised in vivo using the ExAssist helper phage with SOLR strain. The sequencing results showed that this insert was 393 bp in length and contained a ploy (A) tail. In order to obtain total length included ORF, the fragment was amplified by 5'-RACE. The cDNA fragment was cloned into pMD-T18 vector and sequenced. The sequencing results showed that this insert was 569bp in length and contained a single open reading frame (ORF) and it was predicted to encode 136 amino acids. This gene was designated TS2 and was gene of Trichinella spiralis by BLAST searches with the GenBank database.ELM analysis showed that TS2 protein has four N-Arg dibasic convertase (nardilysine) cleavage site,one peptide C-terminal amidation site,one glycosaminoglycan attachment site,DNAStar software analysis in antigenic index showed that TS2 protein has six epitope sites. dot blot hybridization demonstrated that TS2 gene was not from myeloma cell but Trichinella spiralis.
Eukaryotic expression of the TS2 gene The fragment of TS2 ORF was amplified by PCR with designed primers according to TS2. The recombinant plasmids pMD18-T-TS2 was constructed. The ligation products were transformed to competent cells of host strain DH5α. The recombinant plasmids pVAX1-TS2 was constructed and transformed into E.coli Rosetta(DH5α)The recombiant plasmids pVAX1-TS2 was expressed in Hela cell strain. After transfected it into Hela cells, the specific recombinant proteins were detected in supernatant of Hela cell by Western blotting.
Anti-tumor effect of Trichinella spiralis on Sp2/0 myeloma cell in BalB/C mice The antitumor effect of TS2 protein against Sp2/0 myeloma cell was studied after the mice were inoculated with Sp2/0 myeloma cell after the mice intramuscular injected with recombinant plasmid, The results showed that recombinant plasmid couldn’t induce response of cellular immunity in mice. The mice inoculated indicated that the group vaccinated with pVAX1-TS2 elicited CD3+、CD4+ T subtype cells significant higher than that of the groups vaccinated with pVAX1 and PBS. The tumor size and the tumor weight of the group injected with recombinant plasmid of the TS2 were notable smaller than that of the groups injected with control plasmid and PBS. The above results suggested that the TS2 protein have better antitumor effect on Sp2/0 myeloma in mice.
引文
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