Preparation Of Human Malignant Melanoma Ganglioside ScFv Antibody Conjugated Quantum Dot Probe And Its Specific Binding With Human Malignant Melanoma Cells

BackgroundMelanoma, also known as malignant melanoma, comes from cells called melanocytes that are present in the epidermis, is highly aggressive and can quickly spread to the body, it can occur at any parts of the body but mostly in skin, yet Its pathogenesis is not clear, once melanoma spread elsewhere in the body, its mortality rate is very high, there is no effective treatment for metastatic melanoma.However, if melanoma was detected and treated early, it is curable in most instances. In recent years molecular imaging has become a hot spot in researching the occurrence and development of melanoma, by antibodies or other biological molecules which can target the tumor cells. This gives hope to the early diagnosis and treatment for tumors.Anti-melanoma associated antigen antibody can be used as probes for the diagnosis and treatment of melanoma. So far, there are many melanoma-associated antigen were found, and most studies were about the ganglioside and high molecular weight associated antigen. We used the anti-human melanoma ganglioside single chain antibody (GD/ScFvMEL) in this study. ScFv is a new member in gene engineering, its size is only one-sixth of a whole antibody, the size advantage make it clear faster in the blood and normal tissue and easier to penetrate into tumor tissue thus, it has been very widely used in diagnosis, treatment and prevention of tumor.You can observe its specific binding with tumor cells in vivo or vitro, by connecting ScFv with fluorescent dyes. In recent years, quantum dots (QDs), particularly water solubility, high yield of QDs has become a hot spot in the biomedical field. QDs was more stable than conventional organic fluorescent dyes, and it can emit different colors of light simultaneously by using different sizes of QDs. Thus, we can observe the living cells or the target molecules on its surface, and research the biological characteristics of these molecules and their mutual relations in the same time. In the biomedical field, QDs, especially water-soluble, high yield of QDs has become a research hotspot.In this study the anti-human melanoma gaglioside single chain variable fragment antibody (GD/ScFvMEL) gene fragments has been formed a new recombinant with pET28a (+) or pET32a (+) and the protein was exprssed in E. coli. The expression product was purified, and on this based, we combined nano-particles quantum dots to the GD/ScFvMEL to explore its possibilities as a new target molecules in vitro imaging and diagnose of melanoma. This study provides a basis for early detection of melanoma.Objective:The objective of this paper is to generatate the GD/ScFvMEL genes in previous study and clone it into pET28a (+) and pET32a (+) vector, after expression and purification, the purified product was connecte with cadmium telluride quantum dots (QDs) to form GD/ScFvMEL-QDs, and observe this nanoprobe's specifity to human malignant melanoma cells.Methods:1. We designed PCR primers with Nde I, EcoR I and Xho I restriction sites and extracted a small amount of plasmid, refering to the instructions of TIAN biochemical Technology (Beijing) Co., Ltd.. According to the DNA polymerase instructions of TaKaRa company, we got the target gene by PCR. The PCR amplification procedures was:94℃,5min→(94℃,30sec→55℃,30sec→72℃, 1min)×35 cycle→72℃,7min. DNA fragments was recycled and connected with pMD18-T vector in the light of the Takara's Reference.2. The fragment with the T-vector and pET28a (+) were digested simultaneously with Nde I and Xho I, the large fragment and target gene were ligated in vitro designated pET28-GD/ScFvMEL. Refer to TIANGEN's description of DH5a competent cells, the plasmid was transformed into E.coli DH5a competent cells in plate containing 50μg/mL kanamycin. Similarly, The fragment with the T-vector and pET32a (+) were digested with EcoR I and Xho I, the large fragment and the target gene were ligated in vitro designated pET32-GD/ScFvMEL. The plasmid was transformed into E.coli DH5a competent cells in plate containing 50μg/mL ampicillin.3. We condulted the PCR and double restriction enzyme digestion on the two recombinant separatly to confirm the sequence were right. Clones that passed the confirmation tests were subjected to DNA sequencing by the Sunny companies in Shanghai.4. After identified the fragments of the targeted gene sequences in the two recombinant are corrected, the pET28-GD/ScFvMEL and pET32-GD/ScFvMEL were transformed into E.coli BL21(DE3) for expression. We explored the conditions of the greatest yeild of the protein by using SDS-PAGE analysis. Bacterial colonies transformed with the plasmid containing the gene insert were grown in Luria broth (LB) growth medium containing 50μg/mL kanamycin (or ampicillin)at 37℃overnight and agitated at 220 rpm. The cultures were then diluted in 1:100 with fresh LB medium plus 50μg/mL kanamycin, grown to A600=0.6 at 37℃. The expression of protein was induced by addition of isopropyl beta-D-thiogalactopyranoside (IPTG) to a 1mmol/L concentration at 37℃for 4 hours. The cells were harvested in 17 OOOxg centrifugation, and stored frozen at-70℃for later purification or-20℃for SDS-PAGE or Western blotting analysis. Protein samples were analyzed by electrophoresis on a 15% SDS-PAGE under reducing conditions. The gels were stained with Coomassie blue. For western blots, proteins were electrophoretically transferred onto nitrocellulose membranes. The membrane were first blocked by incubation with 5% BSA. After removing the solution, the membrane was rinsed with TBST three times, a process repeatedly executed after each following step. The membrane were incubated with mouse anti-his monoclonal antibody, further incubated with goat anti-mouse peroxidase conjugate (HRP) antibody, and finally developed using the precipitation type of one-component TMB substrate solution.5. Purification of GD/ScFvMEL protein expressionThe purification and refolding procedures were according to Qiangen's Ni-NTA Superflow Cartridges specification. The cell pellet was thawed for 15 min on ice and resuspended in urea buffer B(8 mol/L Urea, 0.1ml/L Tris-Cl, 0.1mol/L NaH2PO4, pH=8.0) at 5 ml per gram wet weight, and stir cells for 15-60 min at room temperature. Extracts were centrifuged at 10,000 g for 30 min. The supernatant was applied to a Ni-NTA Superflow Cartridges equilibrated. After that, the column was wash to remove non-specific binding with the urea buffer C(8 mol/L Urea, 0.1mol/L Tris-Cl, 0.1mol/L NaH2PO4, pH=6.3) and desired proteins were eluted with urea buffer E(8mol/L Urea, 0.1mol/L Tris·Cl, 0.1mol/L NaH2PO4, pH=4.5). The protein was quantitated by using bradford protein assay kit. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses were performed to determine which fraction(s) contained the majority of polyhistidine-tagged (His-tag) protein. Fractions were diluted in the dilution (4mol/L Urea, 0.1mol/L Tris·Cl, 0.1mol/L NaH2PO4, pH 8.5) to a final a final concentration of 0.1mg/ml and dialyzed against refolding bufferⅠ(2 mol/L Urea, 0.1mol/L Tris-HCl (pH 8.5), 0.1mol/L NaH2PO4,2 mmol/L EDTA,50μmol/L CuSO4,0.9 mmol/L GSH, 0.1mmol/L GSSH,0.5 mol/L L-Arginine,) for 12 hours, and then changed the buffer with refolding bufferⅡ(0.1mol/L Tris-HCl (pH 8.5), 0.1mol/L NaH2PO4,2 mmol/L EDTA,50μmol/L CuSO4,0.9 mmol/L GSH, 0.1mmol/L GSSH,0.5mol/L L-Arginine,1 mol/L Urea). At last, we changes dialysis buffer with PBS (pH 7.5) for 24 hours. The final GD/ScFvMEL product was concentrated by a centrifugal filter devices(Millipore) and stored at 4℃.6. CdTe quantum dots were synthesized and provided by the laboratory of Professor Cui Daxiang. The characterization of the QDs were analysed by using high-resolution transmission electron microscopy and fluorescence spectrophotometer.7. The final protein were conjoined to quantum dots designated nano-probe respectively:600μl prepared QDs were actived by 300μl 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the concentration of 2mg/ml respectively for 5 min by ultrasound. Then 300μl target protein in the concentration of 2 mg/ml was added. The solution was allowed to react at room temperature for 2 h on a rotating mixer to obtain pET28a-GD/ScFvMEL-QDs or pET32a-GD/ScFvMEL-QDs. The final nanoprobes was purified by using affinity chromatography to remove unbound quantum dots and stored at 4℃. The prepared nanoprobes was analyzed by fluorescence spectrophotometer and SDS-PAGE electrophoresis.8. Nano-probe (GD/ScFvMEL-QDs) targeting melanoma cells in imaging experiments Maligant melanoma A375 cell line, human gastric cancer MGC-803 cell line were plated on 24-well chamber slides and cultured for 24 h in DEME with 10% (v/v) FCS at 37℃in a humidified 5% CO2 incubator. Cells were fixed in 4% paraformaldehyde for 15 min were blocked with 3% BSA for 10 min, incubated with nano-probe at 4℃overnight. After several washing steps, cells were analyzed under a OlympusⅨ71 fluorescent microscope. Photographs were taken with a scope-mounted camera.Results:1. Cloning of GD/ScFvMEL gene and construction of pET28a-GD/ScFvMEL and pET32a-GD/ScFvMEL GD/ScFvMEL gene fragments were successfully amplified, the amplified target gene fragments and pET28a(+) vector were digested by NdeⅠand XhoⅠ, the GD/ ScFvMEL gene was introduced into the pET28a (+) bacterial expression vector to form pET28a-GD/ScFvMEL. Similarly, the amplified target gene fragments and pET32a(+) vector were digested by EcoRⅠand XhoⅠ, the GD/ScFvMEL gene was introduced into the pET32a(+) bacterial expression vector to form pET32a-GD/ScFvMEL. After PCR and double restriction enzyme digestion(Nde I and Xho I or EcoR I and Xho I) on the two recombinant separatly, the size of the PCR product (729bp) and the fragments (729bp) of the digestion were consistent with the expected results. The recombinant were also confirmed by DNA sequencing.2. Expression and purification of GD/ScFvMEL protein The expression of the GD/ScFvMEL protein was induced by addition of isopropyl beta-D-thiogalactopyranoside (IPTG) to a lmmol/L concentration at 37℃for 7 hours, we took out one sample per hour for SDS-PAGE analysis using non-induced bacilli as a negative control, the results showed that the protein molecular weight was about 29000(pET28a-GD/ScFvMEL) and 45 000(pET32a-GD/ScFvMEL), which was consistent with the expected protein size; The amount of expression of the protein increased with time, in the induction of 4 h, The amount of the target protein expression reached the maximum. The proportion of expressed GD/ScFvMEL antibody in total bacteria proteins was about 40% as detected total proteins by SDS-PAGE. By using anti-His antibody and goat anti-mouse peroxidase conjugate (HRP) antibody, positive bands appeared in the Western blotting result, which demonstrated that the prepared GD /ScFvMEL is specific to to anti-His antibody.3. Characterization of CdTe QDs and identification of the nano-probe High-resolution electron micrograph clearly shows that size of the prepared quantum dots was uniform, and diameter was 5 nm; X diffraction analysis results show that the preparaed quantum dots containing cadmium and telluride elements, which was consistent with expectations. Analysisi of fluorescence spectra show that the fluorescence peak of QDs shifted to the left for 20nm after QDs was conjointed with GD/ScFvMEL, which demonstrate that QDs was successfully marked on the GD/ScFvMEL. Moreover, fluorescence intensity of the labeled quantum dots increased a little, showd that protein enhanced quantum dots with fluorescence signals, which was consistent with some reports. Electrophoresis analysis of the nanoprobes revealed that, GD/ScFvMEL was at 29000 (pET28a-GD/ScFvMEL) and 45000 (pET32a-GD/ScFvMEL),while the nano-probe is located in the sample hole, can not enter the gel, further evidence that The purified-and refolded-GD/ScFvMEL antibody was effectively conjugated with CdTe quantum dots, and the resulting GD/ScFvMEL-QDs nanoprobe was prepared.4. Analysis of the specificity of GD/ScFvMEL-QDs to tumor cell under a fluorescence microscope A375 melanoma cells and gastric cancer MGC-803 cells were incubated with nano-probe, and then we observed the results under a fluorescence microscope. The fluorescence intensity of the comparison experiments, which were designed for the purpose of inspecting the effect of non-specific biocativity of this analysis, was quite low, proving that no observable non-specific adsorption occurred. And the fluorescence intensity of the experiments was very strong. The nanoprobe specifically binded to melanoma A375 cells but not stomach cancer MGC-803 cells. Thus, GD/ScFvMEL-QDs is directly demonstrated to be specific to melanoma cells.Conclusion:1. Through this experiment, we obtain a human source GD/ScFvMEL gene, and was highly expressed for the first time in the pET system;2. A anti-human melanoma gaglioside single-chain antibody-CdTe quantum dot nanoprobe is successfully prepared, and it can specifically bind to melanoma cells, non-binding to control cells, demostrated that the nanoprobes have good specificity and was able to target melanoma cells. This results provied a solid foundation for further study to reserach the antigen binding mechanism of GD/ ScFvMEL, or construct a bifunctional antibodies, as well as further diagnosis of melanoma by molecular imaging.