新型重组蛋白TAT/54R/KDEL的构建和表达及其对CXCR4的表型敲除活性研究
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摘要
研究表明,趋化因子受体CXCR4(CXC chemokine receptor 4)与其特异性配体,基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)相互作用在多种恶性肿瘤转移过程中起着关键作用。因而,CXCR4被认为是有效防治肿瘤转移的重要靶点。本课题组曾对SDF-1进行遗传改造,获得了一种CXCR4竞争性拮抗剂:SDF-1/54R,研究证实:SDF-1/54R可通过诱导CXCR4内吞,使细胞膜表面CXCR4迅速消失。但由于内吞进细胞的CXCR4又回到质膜表面重新利用,导致SDF-1/54R对CXCR4介导细胞迁移的抑制效应也随之消失[1]。因此,欲获得真正具有临床抗肿瘤转移应用价值的CXCR4拮抗剂,尝试持久性阻断CXCR4的技术路线是十分必要的。鉴于胞内因子(intrakine)分子量小,特异性强,靶向性高等优点,将胞内因子表型敲除技术用于持久性阻断肿瘤细胞CXCR4的表达不失为一种新思路。为此,课题组进一步尝试将SDF-1/54R与内质网定位片段KDEL嵌合,从而构建了胞内因子SDF-1/54R/KDEL的真核载体和腺病毒载体。体外实验证实,转染SDF-1/54R/KDEL基因的MOLT-4细胞膜表面CXCR4的表达量显著降低,其转移受到持久性抑制。然而,考虑到转基因技术操作复杂,在整体应用上安全性无法保证等局限,我们有必要进一步尝试直接向胞内递送趋化因子蛋白,在蛋白水平上实现CXCR4表型敲除以抑制肿瘤转移的可能性。为此,本课题借鉴穿膜肽的最新研究成果,再次对SDF-1/54R/KDEL进行基因改造,将其与高效穿膜载体TAT (47~57)连接,从而构建了一种新的嵌合蛋白:TAT/54R/KDEL。拟利用TAT(47~57)的高效穿膜能力和KDEL的内质网定位作用,将CXCR4的特异性拮抗剂SDF-1/54R导入肿瘤细胞,在蛋白水平上实现对其细胞膜表面CXCR4的敲除,进而实现抑制肿瘤细胞转移的目的。本研究结果将为证实这一防治肿瘤转移新策略的可行性和有效性提供直接的实验依据,并为其它以CXCR4为靶点的肿瘤基因治疗提供有益的参考价值。
目的:
对SDF-1/54R进行二次基因改造,将其与高效穿膜载体TAT (47~57)和内质网定位片段KDEL连接,构建一种新的嵌合蛋白:TAT/54R/KDEL,并在细胞水平上证实其CXCR4表型敲除功能以及评价其在体内对肿瘤转移的抑制作用。
方法:
①以本室构建的GFP/pET-28a(+)和54R/pET-28a(+)为模板,采用PCR方法扩增GFP/KDEL和54R/KDEL基因,PCR产物酶切后插入pTAT-HA原核表达载体TAT编码区下游的多克隆位点,即获得含有目的基因的重组质粒TAT/GFP/KDEL/pTAT-HA和TAT/54R/KDEL/pTAT-HA。
②将鉴定正确的重组质粒TAT/GFP/KDEL/pTAT-HA和TAT/54R/KDEL /pTAT-HA转化表达菌株BL21,用0.5μM IPTG诱导表达,表达产物采用Western blot法进行鉴定。并利用表达产物N-端所带的6×His-Tag融合标签,采用镍柱亲和层析和HPLC纯化目的蛋白,由于嵌合蛋白TAT/GFP/KDEL为可溶表达,纯化后的蛋白只需简单的透析、浓缩即可得到有活性的蛋白。而嵌合蛋白TAT/54R/KDEL以包涵体形式表达,纯化后的蛋白需要联合采用稀释、透析和超滤的方法进行复性。
③体外实验,首先检测TAT/GFP/KDEL对MOLT-4细胞的穿膜能力和内质网定位情况以考察本课题设计方案的可行性,然后分别利用荧光显微镜和荧光酶标仪检测TAT/54R/KDEL对高表达CXCR4的MOLT-4细胞和不表达该受体的CNE2细胞的穿膜能力和靶向性,并利用激光共聚焦显微镜检测TAT/54R/KDEL的内质网定位情况。TAT/54R/KDEL的CXCR4表型敲除能力是其体外活性研究的重点,为此,我们首先采用流式细胞仪检测TAT/54R/KDEL对MOLT-4细胞膜表面CXCR4表达量的影响;然后利用趋化实验考察TAT/54R/KDEL对SDF-1诱导的MOLT-4细胞转移的抑制作用;最后通过Western blot实验进一步评价TAT/54R/KDEL对细胞表面CXCR4表型敲除的作用效果。此外,还利用台盼蓝排斥实验和MTT法检测了TAT/54R/KDEL的细胞毒性及其对细胞增殖的影响。
④将高表达CXCR4的小鼠乳腺癌细胞4T1,原位接种于BALB/c小鼠第2对乳房脂肪垫以建立小鼠乳腺癌转移模型,并利用该模型考察TAT/54R/KDEL对移植部位乳腺癌细胞生长和转移的抑制效应。进一步采用RT-PCR和流式细胞仪检测乳腺癌原发灶及转移灶的CXCR4和SDF-1表达量的变化,以探讨TAT/54R/KDEL对乳腺癌转移的抑制效应与其CXCR4表型敲除能力之间的相互关系,从而初步阐明TAT/54R/KDEL在体内的作用机制。
结果:
①以本室构建的GFP/pET-28a(+)和54R/pET-28a(+)为模板,PCR扩增出目的基因GFP/KDEL和54R/KDEL,并采用PCR、酶切和测序方法证实目的基因已正确地插入pTAT-HA载体。
②用Western blot法证实含有目的基因的重组质粒TAT/GFP/KDEL/pTAT-HA和TAT/54R/KDEL/pTAT-HA在BL21菌株中成功实现了高表达,采用镍柱亲和层析和HPLC纯化得到了纯度大于95 %的目的蛋白TAT/GFP/KDEL和TAT/54R/KDEL。TAT/54R/KDEL经稀释、透析和超滤等处理后实现了复性。
③分别利用荧光显微镜、流式细胞仪和荧光酶标仪证实TAT/GFP/KDEL具有高效的穿膜能力,并能准确地定位于内质网,从而初步证实了本课题设计方案的可行性。与不表达CXCR4的CNE2细胞相比,TAT/54R/KDEL对高表达该受体的MOLT-4细胞具有更高的穿膜能力和靶向性,并且也能准确地定位于内质网为其进一步实现CXCR4表型敲除奠定了基础。通过流式细胞术的方法证实TAT/54R/KDEL对MOLT-4细胞表面CXCR4具有持续地表型敲除能力。趋化实验表明TAT/54R/KDEL能够有效地抑制SDF-1诱导的肿瘤细胞迁移,Western blot实验进一步证实与SDF-1/54相比较,TAT/54R/KDEL对肿瘤细胞CXCR4的表型敲除效果更完全。台盼蓝排斥实验和MTT法证实TAT/54R/KDEL没有细胞毒性,不影响CNE2细胞的增殖,而对MOLT-4细胞的增殖具有抑制作用。
④将高表达CXCR4的乳腺癌细胞4T1,原位接种于BALB/c小鼠第2对乳房脂肪垫成功建立了小鼠乳腺癌转移模型,并利用该模型证实TAT/54R/KDEL对移植部位乳腺癌细胞的生长和转移具有抑制效应。进一步采用RT-PCR和流式细胞术证实乳腺癌的转移与细胞膜表面CXCR4及转移组织SDF-1的高表达密切相关,TAT/54R/KDEL通过蛋白水平上CXCR4的表型敲除在一定程度上抑制了乳腺癌的转移。
结论:
①成功构建了TAT/54R/KDEL的大肠杆菌表达系统,并且表达、分离纯化出具有生物活性的TAT/54R/KDEL蛋白。
②TAT/54R/KDEL具有高效的穿膜能力,并能准确地定位于内质网,初步证实了本课题设计方案的可行性。
③体外实验证实TAT/54R/KDEL对MOLT-4细胞膜表面的CXCR4具有持续地表型敲除能力,并且能够有效抑制SDF-1诱导的细胞迁移。
④通过小鼠乳房脂肪垫移植4T1细胞,成功建立了小鼠乳腺癌转移模型。
⑤体内实验证实TAT/54R/KDEL可在一定程度上抑制乳腺癌的转移。
It has been shown that interaction between CXCR4 (CXC chemokine receptor 4) and its specific ligand SDF-1(Stromal cell-derived factor-1) plays an important role in the metastasis of many malignant tumors. Therefore, it may be of great significance to effectively prevent tumor metastasis by targeting CXCR4 and blocking interaction between CXCR4 and SDF-1. Therefore, a competitive antagonist of CXCR4, named SDF-1/54R, was constructed by gene reconstruction of SDF-1 in our previous research, and it has been proved that SDF-1/54R could eliminate CXCR4 on cell surface by induction of internalization of CXCR4 quickly, however, the inhibitory effect of SDF-1/54R on CXCR4 induced cell migration was also temporary because of the re-expression of internalized CXCR4 receptor on cell membrane. Accordingly, a new approach to permanently inhibit CXCR4 expression is necessary in order to obtain a clinical valuable CXCR4 antagon. The intrakine phaenotype knockout technology will be a new strategy to block CXCR4 expression on tumor cell surface permanently due to the properties of intrakine such as low molecular weight, strong specificity and high targetting. Above on this, eukaryotic vector and adenovirus vector of intrakine, SDF-1/54R/KDEL, were constructed by linking SDF-1/54R and an endoplasmic reticulum (ER) retention signal segment (KDEL) together. It has been demonstrated that expression of CXCR4 on MOLT-4 cell which transfected by SDF-1/54R/KDEL was reduced dramatically and a permanent inhibition of migration was observed. Allow for the multiplicity of transgenic technology operation and the low safety ensurance in systemic application, a further strategy of delivering chemotatic factor intracellularly to knockout CXCR4 on cell surface was necessary. Therefore, based on the newest research of transmembrane peptide, the second gene reconstruction of SDF-1/54R/KDEL was carried on by linking a highly effective transmembrane vector TAT(47～57) to its N-extremity. The high transmembrane effect of TAT and the ER retention function of KDEL both help to introduce fusion protein SDF-1/54R into ER of tumor cells in order to specifically bind to the newly-synthetic target receptor CXCR4 intracellularly, accordingly block the expression of CXCR4 on cell surface and consequently the following tumor metastasis signaling mediated by CXCR4/SDF-1 interaction. The results will provide direct experimental evidence to investigate the feasibility and effectivity of the new strategy which exploring CXCR4 as a target in cancer gene therapy.
Objectives:
A new chimeric protein: TAT/54R/KDEL was constructed by the second gene reconstruction of SDF-1/54R, via a highly effective transmembrane vector TAT(47～57) and the endoplasmic reticulum (ER) retention signal (KDEL) linked to each extremity of SDF-1/54R respectively, and the inhibition of tumor metastasis was tested both in vitro and in vivo.
Methods:
①GFP/KDEL and 54R/KDEL gene were obtained from GFP/pET-28a(+) and 54R/pET-28a(+) constructed previously with PCR, and linked with KDEL gene fragment respectively. Then the two part were inserted into the downstream MCS of TAT in prokaryotic expression vector pTAT-HA respectively to obtain the recombinant plasmids TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA .
②The identificated recombinant plasmids TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA were subsequently transformed into E coli expression strain BL21(DE3), the expression of recombinant protein was induced by 0.5μM IPTG. and verificated by Western blot. Then a Ni2+ affinity chromatography column was used to purificate recombinant protein by the 6×His-Tag at the N-extremity of the protein. Active TAT/GFP/KDEL could be obtained just by dislysis and concentration due to its soluble expression property, however, TAT/54R/KDELL needs deliquation, dislysis and ultrafiltration for renaturation as it exists in inclusion body.
③MOLT-4 cell line was used to test the transmembrane and ER retention capability of TAT/GFP/KDEL to determine the feasibility of designed proposal initially. The bioactivity of TAT/54R/KDEL as transmembrane capability and targeting property was detected by fluorescence microscope and fluorescence microplate reader on MOLT-4 cells which have an overexpression of CXCR4 and CNE2 cells which are CXCR4 negative. Laser confocal microscopy was used to detect intracellular location of TAT/54R/KDEL on ER. It is of great importance to test CXCR4 knockout effect of TAT/54R/KDEL, so a flow cytometer was used firstly to detect influence of TAT/54R/KDEL on CXCR4 expression on cell surface, then a chemotaxis essay to investigate TAT/54R/KDEL’s inhibitory effect on MOLT-4 cell migration and finally Western blot was applied to evaluate the effectiveness of TAT/54R/KDEL on CXCR4 knockout on cell surface. Besides, trypan blue rejection experiment and MTT method was used to test cytotoxicity and influence on cell proliferation of TAT/54R/KDEL.
④The animal model of breast cancer metastasis was established by transplanting 4T1 cell into the second breast subcutaneous fat pad of BALB/c mice to detect the inhibitory effect of TAT/54R/KDEL on development and metastasis of the transplanted tumor in tumor-bearing mice. RT-PCR and flow cytometer was applied to detect variation of CXCR4 and SDF-1 expression in primary tumor and metastatic tumor in order to investigate the interaction of metastasis inhibition role of TAT/54R/KDEL and its knockout effect on CXCR4, and illuminate the action mechanism of TAT/54R/KDEL in vivo.
Results:
①GFP/KDEL and 54R/KDEL gene were obtained successfully with PCR from GFP/pET-28a(+) and 54R/pET-28a(+) constructed previously and inserted into pTAT-HA vector. Both were confirmed to be correct by PCR, enzyme digestion and gene sequencing.
②Overexpression of TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA in BL21 was confirmed by Western blot. Purified target proteins TAT/GFP/KDEL and TAT/54R/KDEL (>95%) were obtained by nickel affinity chromatography and HPLC purification. The TAT/54R/KDEL in inclusion body was renaturated by dilution, dislysis and ultrafiltration.
③High transmembrane effectiveness and capability of exact ER retention of TAT/GFP/KDEL in MOLT-4 cells was verified by fluorescence microscope and fluorescence microplate reader, which offering a initial feasibility of the designed proposal. When compared with CXCR4- CNE2 cells, TAT/54R/KDEL showed a better transmembrane and targeting capability, and exact located on ER in MOLT-4 cells on which CXCR4 is overexpressed, which established the foundation of CXCR4 phenotype knockout. Flow cytometry proved that TAT/54R/KDEL had a permanent CXCR4 phenotype knockout effect on MOLT-4 cells. Chemotaxis essay showed that TAT/54R/KDEL could restrain SDF-1 induced cell migration effectively and Western blot demonstrated that the CXCR4 phenotype knockout effectiveness of TAT/54R/KDEL was more thoroughly compared with SDF-1/54. Trypan blue rejection experiment and MTT method confirmed that TAT/54R/KDEL has a proliferation inhibitory effect on MOLT-4 cells which has no effect on CNE2 cells.
④The animal model of breast cancer metastasis was established successfully by transplanting 4T1 cell into the second breast subcutaneous fat pad of BALB/c mice, and a dramatic inhibitory effect of TAT/54R/KDEL on development and metastasis of the transplanted tumor was detected. The correlation breast tumor metastasis with CXCR4 expression on tumor cell surface and SDF-1 expression in metastatic tumor tissue was confirmed to be close by RT-PCR and flow cytometry. TAT/54R/KDEL has a inhibition role on breast cancer metastasis to some extent via its CXCR4 knockout effect.
Conclusion:
①E.Coli expression system of TAT/GFP/KDEL and TAT/54R/KDEL was constructed successfully and bioactive TAT/GFP/KDEL and TAT/54R/KDEL proteins were obtained by expression, separation and purification.
②High transmembrane effectiveness and capability of exact ER retention of TAT/GFP/KDEL on MOLT-4 cells were verified, confirming the feasibility of the designed proposal initially.
③TAT/54R/KDEL has a permanent CXCR4 phenotype knockout effect on MOLT-4 cell surface in vitro.
④The animal model of breast cancer metastasis was established successfully by transplanting 4T1 cell into the breast subcutaneous fat pad of BALB/c mice.
⑤The inhibition role of TAT/54R/KDEL on breast cancer metastasis was confirmed by experiment in vivo to some extent.
目的:
对SDF-1/54R进行二次基因改造,将其与高效穿膜载体TAT (47~57)和内质网定位片段KDEL连接,构建一种新的嵌合蛋白:TAT/54R/KDEL,并在细胞水平上证实其CXCR4表型敲除功能以及评价其在体内对肿瘤转移的抑制作用。
方法:
①以本室构建的GFP/pET-28a(+)和54R/pET-28a(+)为模板,采用PCR方法扩增GFP/KDEL和54R/KDEL基因,PCR产物酶切后插入pTAT-HA原核表达载体TAT编码区下游的多克隆位点,即获得含有目的基因的重组质粒TAT/GFP/KDEL/pTAT-HA和TAT/54R/KDEL/pTAT-HA。
②将鉴定正确的重组质粒TAT/GFP/KDEL/pTAT-HA和TAT/54R/KDEL /pTAT-HA转化表达菌株BL21,用0.5μM IPTG诱导表达,表达产物采用Western blot法进行鉴定。并利用表达产物N-端所带的6×His-Tag融合标签,采用镍柱亲和层析和HPLC纯化目的蛋白,由于嵌合蛋白TAT/GFP/KDEL为可溶表达,纯化后的蛋白只需简单的透析、浓缩即可得到有活性的蛋白。而嵌合蛋白TAT/54R/KDEL以包涵体形式表达,纯化后的蛋白需要联合采用稀释、透析和超滤的方法进行复性。
③体外实验,首先检测TAT/GFP/KDEL对MOLT-4细胞的穿膜能力和内质网定位情况以考察本课题设计方案的可行性,然后分别利用荧光显微镜和荧光酶标仪检测TAT/54R/KDEL对高表达CXCR4的MOLT-4细胞和不表达该受体的CNE2细胞的穿膜能力和靶向性,并利用激光共聚焦显微镜检测TAT/54R/KDEL的内质网定位情况。TAT/54R/KDEL的CXCR4表型敲除能力是其体外活性研究的重点,为此,我们首先采用流式细胞仪检测TAT/54R/KDEL对MOLT-4细胞膜表面CXCR4表达量的影响;然后利用趋化实验考察TAT/54R/KDEL对SDF-1诱导的MOLT-4细胞转移的抑制作用;最后通过Western blot实验进一步评价TAT/54R/KDEL对细胞表面CXCR4表型敲除的作用效果。此外,还利用台盼蓝排斥实验和MTT法检测了TAT/54R/KDEL的细胞毒性及其对细胞增殖的影响。
④将高表达CXCR4的小鼠乳腺癌细胞4T1,原位接种于BALB/c小鼠第2对乳房脂肪垫以建立小鼠乳腺癌转移模型,并利用该模型考察TAT/54R/KDEL对移植部位乳腺癌细胞生长和转移的抑制效应。进一步采用RT-PCR和流式细胞仪检测乳腺癌原发灶及转移灶的CXCR4和SDF-1表达量的变化,以探讨TAT/54R/KDEL对乳腺癌转移的抑制效应与其CXCR4表型敲除能力之间的相互关系,从而初步阐明TAT/54R/KDEL在体内的作用机制。
结果:
①以本室构建的GFP/pET-28a(+)和54R/pET-28a(+)为模板,PCR扩增出目的基因GFP/KDEL和54R/KDEL,并采用PCR、酶切和测序方法证实目的基因已正确地插入pTAT-HA载体。
②用Western blot法证实含有目的基因的重组质粒TAT/GFP/KDEL/pTAT-HA和TAT/54R/KDEL/pTAT-HA在BL21菌株中成功实现了高表达,采用镍柱亲和层析和HPLC纯化得到了纯度大于95 %的目的蛋白TAT/GFP/KDEL和TAT/54R/KDEL。TAT/54R/KDEL经稀释、透析和超滤等处理后实现了复性。
③分别利用荧光显微镜、流式细胞仪和荧光酶标仪证实TAT/GFP/KDEL具有高效的穿膜能力,并能准确地定位于内质网,从而初步证实了本课题设计方案的可行性。与不表达CXCR4的CNE2细胞相比,TAT/54R/KDEL对高表达该受体的MOLT-4细胞具有更高的穿膜能力和靶向性,并且也能准确地定位于内质网为其进一步实现CXCR4表型敲除奠定了基础。通过流式细胞术的方法证实TAT/54R/KDEL对MOLT-4细胞表面CXCR4具有持续地表型敲除能力。趋化实验表明TAT/54R/KDEL能够有效地抑制SDF-1诱导的肿瘤细胞迁移,Western blot实验进一步证实与SDF-1/54相比较,TAT/54R/KDEL对肿瘤细胞CXCR4的表型敲除效果更完全。台盼蓝排斥实验和MTT法证实TAT/54R/KDEL没有细胞毒性,不影响CNE2细胞的增殖,而对MOLT-4细胞的增殖具有抑制作用。
④将高表达CXCR4的乳腺癌细胞4T1,原位接种于BALB/c小鼠第2对乳房脂肪垫成功建立了小鼠乳腺癌转移模型,并利用该模型证实TAT/54R/KDEL对移植部位乳腺癌细胞的生长和转移具有抑制效应。进一步采用RT-PCR和流式细胞术证实乳腺癌的转移与细胞膜表面CXCR4及转移组织SDF-1的高表达密切相关,TAT/54R/KDEL通过蛋白水平上CXCR4的表型敲除在一定程度上抑制了乳腺癌的转移。
结论:
①成功构建了TAT/54R/KDEL的大肠杆菌表达系统,并且表达、分离纯化出具有生物活性的TAT/54R/KDEL蛋白。
②TAT/54R/KDEL具有高效的穿膜能力,并能准确地定位于内质网,初步证实了本课题设计方案的可行性。
③体外实验证实TAT/54R/KDEL对MOLT-4细胞膜表面的CXCR4具有持续地表型敲除能力,并且能够有效抑制SDF-1诱导的细胞迁移。
④通过小鼠乳房脂肪垫移植4T1细胞,成功建立了小鼠乳腺癌转移模型。
⑤体内实验证实TAT/54R/KDEL可在一定程度上抑制乳腺癌的转移。
It has been shown that interaction between CXCR4 (CXC chemokine receptor 4) and its specific ligand SDF-1(Stromal cell-derived factor-1) plays an important role in the metastasis of many malignant tumors. Therefore, it may be of great significance to effectively prevent tumor metastasis by targeting CXCR4 and blocking interaction between CXCR4 and SDF-1. Therefore, a competitive antagonist of CXCR4, named SDF-1/54R, was constructed by gene reconstruction of SDF-1 in our previous research, and it has been proved that SDF-1/54R could eliminate CXCR4 on cell surface by induction of internalization of CXCR4 quickly, however, the inhibitory effect of SDF-1/54R on CXCR4 induced cell migration was also temporary because of the re-expression of internalized CXCR4 receptor on cell membrane. Accordingly, a new approach to permanently inhibit CXCR4 expression is necessary in order to obtain a clinical valuable CXCR4 antagon. The intrakine phaenotype knockout technology will be a new strategy to block CXCR4 expression on tumor cell surface permanently due to the properties of intrakine such as low molecular weight, strong specificity and high targetting. Above on this, eukaryotic vector and adenovirus vector of intrakine, SDF-1/54R/KDEL, were constructed by linking SDF-1/54R and an endoplasmic reticulum (ER) retention signal segment (KDEL) together. It has been demonstrated that expression of CXCR4 on MOLT-4 cell which transfected by SDF-1/54R/KDEL was reduced dramatically and a permanent inhibition of migration was observed. Allow for the multiplicity of transgenic technology operation and the low safety ensurance in systemic application, a further strategy of delivering chemotatic factor intracellularly to knockout CXCR4 on cell surface was necessary. Therefore, based on the newest research of transmembrane peptide, the second gene reconstruction of SDF-1/54R/KDEL was carried on by linking a highly effective transmembrane vector TAT(47～57) to its N-extremity. The high transmembrane effect of TAT and the ER retention function of KDEL both help to introduce fusion protein SDF-1/54R into ER of tumor cells in order to specifically bind to the newly-synthetic target receptor CXCR4 intracellularly, accordingly block the expression of CXCR4 on cell surface and consequently the following tumor metastasis signaling mediated by CXCR4/SDF-1 interaction. The results will provide direct experimental evidence to investigate the feasibility and effectivity of the new strategy which exploring CXCR4 as a target in cancer gene therapy.
Objectives:
A new chimeric protein: TAT/54R/KDEL was constructed by the second gene reconstruction of SDF-1/54R, via a highly effective transmembrane vector TAT(47～57) and the endoplasmic reticulum (ER) retention signal (KDEL) linked to each extremity of SDF-1/54R respectively, and the inhibition of tumor metastasis was tested both in vitro and in vivo.
Methods:
①GFP/KDEL and 54R/KDEL gene were obtained from GFP/pET-28a(+) and 54R/pET-28a(+) constructed previously with PCR, and linked with KDEL gene fragment respectively. Then the two part were inserted into the downstream MCS of TAT in prokaryotic expression vector pTAT-HA respectively to obtain the recombinant plasmids TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA .
②The identificated recombinant plasmids TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA were subsequently transformed into E coli expression strain BL21(DE3), the expression of recombinant protein was induced by 0.5μM IPTG. and verificated by Western blot. Then a Ni2+ affinity chromatography column was used to purificate recombinant protein by the 6×His-Tag at the N-extremity of the protein. Active TAT/GFP/KDEL could be obtained just by dislysis and concentration due to its soluble expression property, however, TAT/54R/KDELL needs deliquation, dislysis and ultrafiltration for renaturation as it exists in inclusion body.
③MOLT-4 cell line was used to test the transmembrane and ER retention capability of TAT/GFP/KDEL to determine the feasibility of designed proposal initially. The bioactivity of TAT/54R/KDEL as transmembrane capability and targeting property was detected by fluorescence microscope and fluorescence microplate reader on MOLT-4 cells which have an overexpression of CXCR4 and CNE2 cells which are CXCR4 negative. Laser confocal microscopy was used to detect intracellular location of TAT/54R/KDEL on ER. It is of great importance to test CXCR4 knockout effect of TAT/54R/KDEL, so a flow cytometer was used firstly to detect influence of TAT/54R/KDEL on CXCR4 expression on cell surface, then a chemotaxis essay to investigate TAT/54R/KDEL’s inhibitory effect on MOLT-4 cell migration and finally Western blot was applied to evaluate the effectiveness of TAT/54R/KDEL on CXCR4 knockout on cell surface. Besides, trypan blue rejection experiment and MTT method was used to test cytotoxicity and influence on cell proliferation of TAT/54R/KDEL.
④The animal model of breast cancer metastasis was established by transplanting 4T1 cell into the second breast subcutaneous fat pad of BALB/c mice to detect the inhibitory effect of TAT/54R/KDEL on development and metastasis of the transplanted tumor in tumor-bearing mice. RT-PCR and flow cytometer was applied to detect variation of CXCR4 and SDF-1 expression in primary tumor and metastatic tumor in order to investigate the interaction of metastasis inhibition role of TAT/54R/KDEL and its knockout effect on CXCR4, and illuminate the action mechanism of TAT/54R/KDEL in vivo.
Results:
①GFP/KDEL and 54R/KDEL gene were obtained successfully with PCR from GFP/pET-28a(+) and 54R/pET-28a(+) constructed previously and inserted into pTAT-HA vector. Both were confirmed to be correct by PCR, enzyme digestion and gene sequencing.
②Overexpression of TAT/GFP/KDEL/pTAT-HA and TAT/54R/KDEL/pTAT-HA in BL21 was confirmed by Western blot. Purified target proteins TAT/GFP/KDEL and TAT/54R/KDEL (>95%) were obtained by nickel affinity chromatography and HPLC purification. The TAT/54R/KDEL in inclusion body was renaturated by dilution, dislysis and ultrafiltration.
③High transmembrane effectiveness and capability of exact ER retention of TAT/GFP/KDEL in MOLT-4 cells was verified by fluorescence microscope and fluorescence microplate reader, which offering a initial feasibility of the designed proposal. When compared with CXCR4- CNE2 cells, TAT/54R/KDEL showed a better transmembrane and targeting capability, and exact located on ER in MOLT-4 cells on which CXCR4 is overexpressed, which established the foundation of CXCR4 phenotype knockout. Flow cytometry proved that TAT/54R/KDEL had a permanent CXCR4 phenotype knockout effect on MOLT-4 cells. Chemotaxis essay showed that TAT/54R/KDEL could restrain SDF-1 induced cell migration effectively and Western blot demonstrated that the CXCR4 phenotype knockout effectiveness of TAT/54R/KDEL was more thoroughly compared with SDF-1/54. Trypan blue rejection experiment and MTT method confirmed that TAT/54R/KDEL has a proliferation inhibitory effect on MOLT-4 cells which has no effect on CNE2 cells.
④The animal model of breast cancer metastasis was established successfully by transplanting 4T1 cell into the second breast subcutaneous fat pad of BALB/c mice, and a dramatic inhibitory effect of TAT/54R/KDEL on development and metastasis of the transplanted tumor was detected. The correlation breast tumor metastasis with CXCR4 expression on tumor cell surface and SDF-1 expression in metastatic tumor tissue was confirmed to be close by RT-PCR and flow cytometry. TAT/54R/KDEL has a inhibition role on breast cancer metastasis to some extent via its CXCR4 knockout effect.
Conclusion:
①E.Coli expression system of TAT/GFP/KDEL and TAT/54R/KDEL was constructed successfully and bioactive TAT/GFP/KDEL and TAT/54R/KDEL proteins were obtained by expression, separation and purification.
②High transmembrane effectiveness and capability of exact ER retention of TAT/GFP/KDEL on MOLT-4 cells were verified, confirming the feasibility of the designed proposal initially.
③TAT/54R/KDEL has a permanent CXCR4 phenotype knockout effect on MOLT-4 cell surface in vitro.
④The animal model of breast cancer metastasis was established successfully by transplanting 4T1 cell into the breast subcutaneous fat pad of BALB/c mice.
⑤The inhibition role of TAT/54R/KDEL on breast cancer metastasis was confirmed by experiment in vivo to some extent.
引文
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