Cloning, Prokaryotic Expression, Purification And Preparation Of Human Ferritin Light Chain Polyclonal Antibody

Lung cancer is the most common malignant tumor worldwide and its incidence and mortality have increased significantly in past decades, which has been severely threatening human health.Tremendous efforts have been put into the identification of cancer-related molecules, which are potentially used as biomarkers for early diagnosis and prognosis of lung cancer. At present, the serum biomarkers commonly used in clinical diagnosis of lung cancer have lower specificity and sensitivity, which have limitations in improvement of early diagnosis of lung cancer. Thus, employment of these biomarkers is not enough for clinical improvement of patients'life quality and prolongation of their survival. Obviously, it is very urgent to find specific and effective biomarkers for lung cancer diagnosis.Previous studies have revealed that abnormal expression of multiple genes is associated with carcinogenesis and development of lung cancer. Ferritin light chain (FTL) is one of these genes.FTL is a subunit of Ferritin,a highly-conserved biological protein with multiple functions and ubiquitously expressed in a variety of organisms.Ferritin is the major intracellular storage of iron that is required for normal cell growth and proliferation. However, excessive iron is potentially harmful since it can catalyse the formation of toxic reactive oxygen species (ROS) via Fenton reaction. In this respect, ferritin has been shown to protect cells against oxidative damage by modulating iron homeostasis and is also involved in immunity processes. At present, the association of oxidative damage of DNA with carcinogenesis of tumors has been defined. Therefore, more attentions have been paid to the relationship between ferritin and tumors. Mammalian ferritins are usually composed of 24 subunits of two different types,termed light chain(L,19,500 Da) and heavy chain (H ,21,000 Da) that have 55% amino acid sequence identity.The ferritin H subunit has a potent ferroxidase activity that catalyses the oxidation of ferrous iron, whereas ferritin L plays a role in iron nucleation and protein stability. Given that there is a close relationship between Ferritin heavy chain and tumor, whether Ferritin light chain, which have high amino acid sequence identity with heavy chain, exhibits similar function remains to be clarified.Recent studies have demonstrated that abnormal expression of FTL gene occurs in many kinds of turmors, such as liver cancer and gastric cancer. Our previous studies with comparative proteome have shown that FTL gene was also expressed in lung cancer, implying that FTL might play an important role in the process of carcinogenesis and progress of lung cancer.In this study, the cloning, expression and purification of human FTL in vitro have been performed. Anti-FTL polyclonal antibody has been prepared. Specifically, these studies were conducted as follows: The full-length cDNA sequence of human FTL has been acquired by RT-PCR technology.Gene recombination technology was performed to ligate FTL gene acquired by Polymerase Chain Reaction to prokaryotic expression vector pET-30a(+), the recombinant plasmid was then transformed into E.coli BL21 and the positive clones were identified by restriction enzyme digestion and PCR. Expression was induced with IPTG of different concentrations for different time and the fusion protein was purified using affinity chromatography, then the purity and content of target protein were detected by SDS-PAGE and Bradford method. Rabbits were immunized with the purified FTL protein and the antiserum was obtained. The specificity of the antibodies was measured by Western blotting.The objective of this study is to construct a prokaryotic expressing vector encoding human FTL gene and grow it in E.coli BL21.Then, to obtain anti-FTL polyclonal antibody by immunizing rabbits with purified fusion protein. This is to provide the tool for subsequent studies on the biological mechanism of FTL and its expression condition in lung cancer tissues.Materials and methods:1 .RNA extraction and amplification of FTL gene by RT-PCRThe total RNA was extracted from specimens of human lung cancer tissues using Trizol.cDNA was synthesed with random primer by using total RNA as the template. Then, with the cDNA as template,FTL was amplified by polymerase chain reaction(PCR) using the pair of special primers designed according to the cDNA sequence providing by NCBI.Semi-quantitive analysis was utilized to compare the expression level of FTL mRNA in lung cancer and in control tissue.The PCR product was separated by 1.0% agarose electrophoresis.2.Construction, transformation and characterization of the prokaryotic expression vector pET30a(+)-FTLThe amplification product of FTL gene and the prokaryotic expression vector pET-30a(+) were digested by EcoR I and Xho I respectively, and the gene fragments were recycled after 1.0% agarose electrophoresis.FTL gene were ligated to pET-30a(+) by the T4 DNA Ligase at proper ratio.The recombinants were transformated into E. coli JM109, and the positive clones were selected using kanamycin resistance, restriction enzyme digestion and PCR amplification. The gene on the plasmid was sequenced, and then the result was compared with the sequence in GeneBank with the software BLAST. 3.Transformation into BL21 of recombination plasmid pET30a(+)-FTL and characterizationThe recombinants with the correct gene fragments by sequence analyis were transformed into E.coli BL21 and the result was identified with the methods aforementioned. 4.Induction expression of FTL fusion protein and expression form analysis and purificationFusion protein expression was induced by IPTG of different concentrations for different time. The culture product was collected and analyzed through SDS-PAGE after denaturation treatment and the expression level under different induction condition was compared. The induced product was treated by ultrosonic equipment and centrifuged, and then the quantiy of target protein in supernatants and pellets were compared by SDS-PAGE.The expression products were purified with Affinity chromatography. 5.Preparation of anti-FTL polyclonal antibodyRabbits of 3～4 months were immunlized with the mixture of purified protein and Freund's Adjuvant.Subcutaneous injection of multiple places was performed.After immunization for 3 times, the rabbit blood was collected and serum was separated. Two-dimensional diffusion method was utilized to measure the titer of antibody. Western blot analysis was performed to detect the reaction between serum and induced target protein,protein extracted from human lung adenocarcinoma tissue and human lung squamous carcinoma tissue. Results:1 .Extraction of total RNA and Amplification of human FTL geneThe total RNA was extracted from human lung cancer tissue. The electrophoresis results showed that there were three strands,which were 28S,18S and 5S.The ratio of A260/A280 exceeded 1.8 by detecting using UV spectrometer, then the RNA was transcripted reversely into cDNA and FTL gene was amplificated with cDNA as the template.The results of agarose electrophoresis showed that a single strand of 528bp was obtained.Semi-quantitive analysis showed that the expression level of FTL mRNA in lung cancer tissue was higher than control,and there was a significant difference between them(P=0.001). Meanwhile,there was no statistical difference of FTL mRNA between human lung squamous carcinoma tissue and human lung adenocarcinoma tissue(P=0.656).2.Construction of recombinant pET30a(+)-FTL and identificationAfter transformation of recombinant pET30a(+)-FTL into JM109,the transformed bacteria were uniformly seeded on the culture plates.After 12—16 hours'culture,about 10 colonies presented on the plate with recombined vector,fewer than the positive control plate,and there were none colonies on the negative control plate.A single colony was selected on the transformed plate for further culture ,then from which plasmids were extracted.The recombinant vectors were digested by the single restriction endoenzyme EcoR I and both EcoR I and Xho I . The results of enzyme digestion revealed that a 5.95kb fragment occurred after EcoR I digestion and two fragments of 5.42kb and 0.528kb occurred after double enzyme digestion. PCR amplification electrophoresis, which used the plasmids as the templates, showed that a gene fragment of 528bp was acquired. The sequence was 99% identical by analyzing with BLAST software.3.Transformation of recombinant plasmid pET30a(+)-FTL into BL21 and verificationWhen recombinatant vectors were tranformed into BL21 using the same identification method, the similar results were obtained as described previously.4.Expression of pET30a(+)-FTL,solubility analysis and protein purificationThe SDS-PAGE results showed that a 25.1kDa protein strand presented on every lane which representing different induction conditions. The position of this band was comparable to that of the expected FTL fusion protein molecular weight. The highest yield of expression protein was achieved when induced with 0.03mmol/L IPTG for 5 hours. The results of solubility analysis indicated that the level of fusion protein in inclusion bodies was about 9.6 times higher than control.More purified protein was obtained by Affinity chromatography.5.Preparation result of anti-FTL antiserumThe results of two-dimensional diffusion showed the existence of antibody generated with a titer of up to 1:64. Western blot results indicated that this antibody could react with the proteins from human lung adenocarcinoma tissue, human lung squamous carcinoma tissue or inducted expression protein. The reaction site of tissue protein was about 19.5kDa, whereas the reaction strand of expression protein was 25.1kDa or so, indicating that this was the anti-FTL polycolonal antibody.Conclusions:1 .The expression level of FTL mRNA in lung cancer tissue was higher than control.2.Prokaryotic expression vector pET30a(+)-FTL was constructed successfully and it couldexpress effeciently.3.By using Ni Affinity chromatography, an effective method for purifying protein with 6-histag, we have obtained highly purified protein.4.Anti-FTL polyclonal antibody was acquired, which provides the powerful tool for verifyingwhether it is the effective biomarker of lung cancer in future research.