Single-chain variable fragment (SCFV) mouse antibody specific to saposin-like protein 2 of the liver fluke fasciola gigantica

Abstract

A recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica. The production process involves; generating a murine antibody library that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica, selecting candidate recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 from the antibody library, and, assessment of antibody properties and specificity, which, in turn, resulting in 6 clones of recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica, namely: scFvrFgSAP2 A9, scFvrFgSAP2 B6, scFvrFgSAP2 C1, scFvrFgSAP2 E8, scFvrFgSAP2 F3 and scFvrFgSAP2 G6.

Description

[0001] SINGLE-CHAIN VARIABLE FRAGMENT (SCFV) MOUSE ANTIBODY SPECIFIC TO

[0002] SAPOSIN-LIKE PROTEIN 2 OF THE LIVER FLUKE FASCIOLA GIGANTICA

[0003] Technical Field

[0004] The present invention relates to the field of medical science, in particular, to the recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica.

[0005] Background Art

[0006] Fasciolosis is a disease caused by infections with the liver flukes, Fasciola gigantica and Fasciola hepatica. In Thailand and Southeast Asian countries, Fasciola gigantica are the predominant cause of fasciolosis. This infection leads to anemia, chronic inflammation, bile duct obstruction, cholangitis, and liver abscesses in infected patients or livestock. Fasciolosis has been observed with high morbidity and mortality rates, as well as significant economic losses in the livestock industry (Richard L et al., 2021. Pathogenicity and virulence of the liver flukes Fasciola hepatica and Fasciola gigantica that cause the zoonosis Fasciolosis / VIRULENCE / 2839-2867). Liver flukes obtain nutrients from the infected host or animal by using saposin-like protein 2 to bind to the surface of red blood cells, resulting in perforation and hemolysis. Subsequently, the flukes release protease enzymes to digest the nutrients (Rudi G et al., 2006. The saposin-like proteins 1, 2, and 3 of Fasciola gigantica! Molecular & Biochemical Parasitology 133-143). This highlights the importance of saposin-like protein 2 in the life cycle of liver flukes. Additionally, this protein can be detected at the early stages of infection, which prompts the researchers to develop a single-chain variable fragments (scFv) that specifically bind to saposin-like protein 2 using genetic engineering techniques. This process cannot occur naturally. Currently, the production of single-chain variable fragments (scFv) using genetic engineering techniques is highly popular. This method reduces the use of laboratory animals, has lower production costs compared to antibody production using hybridoma techniques, allows for effective quality control of antibodies in each production cycle, and can be scaled up easily by cultivating bacterial or animal cells for industrial production. Hence, this method can reduce the production costs of lower the cost of diagnostic test kits and other technologies that utilize antibody fragments.

[0007] Accordingly, such genetic engineering technique involves randomly combined the variable regions of both the heavy and light chains of antibodies using a linker peptide to create an antibody library in vitro. The resulting antibody library can have a diversity ranging from 106to 108for small libraries, and from 109to 1012for large libraries, which cannot occur naturally. The antibody library is then expressed on the minor coat protein (pill) of a phage for selecting candidate fragment that specifically bind to the target antigen with high affinity (biopanning). This method is highly popular as it can be used in both basic and advanced research and is applicable in various biotechnology industries. (Nina E.W. Christopher J.H. 2009. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnology Advances, 502-520; Philipp H. Peter J.H. 2005. Engineered antibody fragments and the rise of single domains. Nature Biotechnology, page 1126-1136)

[0008] Summary of Invention

[0009] The recombinant murine single-chain variable fragment ( scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica. The production process involves; generating a murine antibody library that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica using advanced genetic engineering technique, selecting candidate recombinant murine single-chain variable fragment ( scFv) that specifically binds to saposin-like protein 2 from the antibody library, and assessment of antibody properties and specificity. Following the abovementioned process, 6 clones of recombinant murine single-chain variable fragment ( scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica, of approximately 30 kilodaltons in molecular weight, were obtained, namely: scFvrFgSAP2 A9, scFvrFgSAP2 B6, scFvrFgSAP2 Cl, scFvrFgSAP2 E8, scFvrFgSAP2 F3 and scFvrFgSAP2 G6.

[0010] The objective of the present invention is to produce the recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica and develop into a diagnostic test for liver fluke infection (fasciolosis) either as an enzyme-linked immunosorbent assay (ELISA) or as an immunochromatographic strip tests (IC strip tests). Additionally, it can be further optimized through antibody engineering to meet the objectives for other applications in the future.

[0011] Description of the Invention

[0012] The production process of the recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica is as follows: A. Generation of a murine antibody library that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica using advanced genetic engineering technique.

[0013] Step 1 Isolation of genes used in antibody production

[0014] The spleen of Balb / c mice was homogenized in 500 pL of TRIzol™ Reagent (Invitrogen) using a homogenizer. Then, 100 pL of chloroform was added into the tissue homogenate. The mixture was mixed thoroughly and incubated at room temperature for 3 minutes. The aqueous phase was separated by centrifugation at 12,000 rpm for 15 minutes at 4°C and mixed with 250 pL of isopropanol. The mixture was next incubated at at -20°C for 12 hours in a freezer. Total RNA was pelleted by centrifugation at 12,000 rpm for 10 minutes at 4°C. Next, the total RNA was washed with 500 pL of 75% (v / v) ethanol and centrifuged at 7,500 rpm for 5 minutes at 4°C. The 75 % (v / v) ethanol was disarded. Total RNA was then air-dried for 10 minutes before rehydrated with 50 pL of nuclease free water. RNA concentration was determined by using a spectrophotometer at 260 nm / 280 nm. RNA was further purified using the RNeasy® Mini Kit (QIAGEN).

[0015] Step 2 cDNA synthesis by reverse transcription reaction cDNA was synthesized from the purified total RNA prepared in the step 1 using the SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen). For the heavy chain, 5 pg of total RNA was mixed with 50 ng of MuIgGl / 2 reverse primer and MuIgG3 reverse primer. For the light chain, 5 pg of total RNA was mixed with MuCK reverse primer and 1 pL of 10 mM dNTP mix. The reaction volume was then adjusted to 10 pL with nuclease- free water. Total RNA / primer mixture was incubated at 65°C for 5 minutes and placed on ice for approximately 2 minutes. Reaction mixture was prepared with 2 pL of 10X RT buffer, 4 pL of 25 m MgCh, 2 pL of 0.1 M DTT, 1 pL of RNaseOUT™ (40 U / pl) and 1 pL of SuperScript™ III RT (200 U / pl). The reaction mixture was rhen added into the total RNA / primer mixture and incubated at 50°C for 50 minutes. The reaction was terminated at 85C for 5 minutes. The RNA template was degraded by adding 1 pL of RNase H and incubated at 37°C for 20 minutes. The cDNA was either used in a PCR reaction or stored at -20°C.

[0016] Step 3 Amplification of antibody heavy and light chain genes from the cDNA using polymerase chain reaction

[0017] In this step, the variable regions of the immunoglobulin heavy chains (VH) and light chains (VL) genes for each antibody family were individually amplified from the cDNA obtained in Step 2 using reverse transcription reaction and primers set specifically designed for each immunoglobulin gene. For the heavy chain, each VH family-specific forward primer (VHI- VH15 / 2) was paired with a reverse primer mixture (JH1-JH ), resulting in 16 separate reactions. For the light chain, each VL family-specific forward primer (VK1-VK13) was paired with a reverse primer mixture (JK1-JK5), resulting in 13 separate reactions. Each 50 pL reaction was composed of 10 pL of 5X SuperFi™ II Buffer, 0.2 rnM dNTP mix, 0.5 pM of each individual forward primer, 0.5 pM of the corresponding reverse primer mix, 100 ng of cDNA, and 1 pL of Platinum™ SuperFi™ II DNA Polymerase. The thermal cycler settings were as follows: 98°C for 1 minute, followed by 35 cycles of 98°C for 10 seconds, 60°C for 10 seconds, and 72°C for 20 seconds, with a final extension at 72°C for 5 minutes and hold at 4°C. PCR products were analyzed using 1.5% (w / v) agarose gel electrophoresis. The bands corresponding to VH (340 bp) and VL (320 bp) genes were excised, purified using the QIAquick® Gel Extraction Kit (QIAGEN), and quantified using a spectrophotometer at 260 nm and 280 nm before future use.

[0018] Step 4 Assembly of the heavy chains and light chains for antibody production

[0019] In this step, 10 ng / pL of the VH DNA mix from individual VH1-VH15 / 2 family, and 10 ng / pL of the VH DNA mix from individual VK1-VK13 family, generated in the step 3 were mixed. The VH DNA mix and VL DNA mix were then linked together using a linker DNA to create a single-chain variable fragment (scFv) antibody gene library through PCR. Each 100 pL reaction was composed of 10 pL of 5X SuperFi™ II Buffer, 0.2 rnM dNTP mix, 0.5 pM scFv-.s / / I forward primer, 0.5 pM scFv-.s / / I reverse primer, 10 ng of VH DNA mix, 10 ng of VL DNA mix and 2 pL of Platinum™ SuperFi™ II DNA Polymerase. The thermal cycler settings were as follows: 98°C for 1 minute, followed by 25 cycles of 98°C for 10 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 5 minutes and hold at 4°C. PCR products were analyzed using 1.5% (w / v) agarose gel electrophoresis. The bands corresponding to scFv antibody gene library approximately 750-800 bp in size were excised, purified using the QIAquick® Gel Extraction Kit (QIAGEN), and quantified using a spectrophotometer at 260 nm and 280 nm before future use.

[0020] Step 5 Digestion of the scFv DNA library with restriction enzymes

[0021] In this step, the 5’ ends of the scFv DNA library generated in the step 4 were digested with the restriction enzyme Sfil. The 40 pL reaction was composed of IX reaction buffer, 2 pg of the scFv DNA library and 40 weiss units of the Sfil enzyme. The digestion reaction was incubated at 50°C for 16 hours. Then, the / / / / [-digested scFv DNA library was purified from the reaction mixture using the QIAquick® PCR Cleanup Kit (QIAGEN). The purified S / H-digested scFv DNA library was then digested with the restriction enzyme Noil to remove the 3' ends of the scFv DNA library. The reaction mixture was adjusted to 30 pL reaction, containing IX reaction buffer and 30 Weiss units of the Noil enzyme, and incubated at 37°C for 16 hours. Subsequently, the Sfil- Notl digested scFv DNA library was separated on a 1.5% (w / v) agarose gel electrophoresis. The bands corresponding to the Sfil-Notl digested scFv DNA library approximately 750-800 bp in size were excised, purified using the QIAquick® Gel Extraction Kit (QIAGEN), and quantified using a spectrophotometer at 260 nm and 280 nm before future use.

[0022] Step 6 Ligation of the scFv DNA library into a phagemid vector

[0023] In this ste, a recombinant vector was generated by ligating the Sfil-Notl digested pCANTAB-5E phagemid vector and the Sfil-Notl digested scFv DNA library generated in the step 5 together at the molar ratio of 2:1. The 20 pL reaction was composed of IX ligation buffer, 200 ng of the Sfil-Notl digested pCANTAB-5E phagemid vector, 100 ng of the Sfil-Notl digested scFv DNA library and 6 weiss units of the T4 DNA ligase enzyme. The ligation reaction was incubated at 4°C for 16 hours. The reaction was terminated at 70°C for 10 minutes and then transformed into Escherichia coli.

[0024] Step 7 Transformation of the recombinant vector into Escherichia coli strain TGI

[0025] In this step, the recombinant vector generated in the step 6 was transformed into Escherichia coli strain TGI cells by heat-shock technique. The Escherichia coli strain TGI were first plated on M9 agar and incubated at 37 °C for 48 hours. A single bacterial colony was then picked and inoculated into SOC medium, where it was cultured until the optical density reached 0.4-0.5, indicating that the bacteria were in the log phase of growth. The culture was then centrifuged at 4,000 rpm for 10 minutes at 4 °C. The pellet was resuspended in the Champion® buffer to obtain chemically competent E. coli TGI cells. Fifty microliters of the chemically competent E. coli TGI cells were aliquoted into 20 tubes, each mixed with 1 pL of the ligated reaction. The tubes were incubated on ice for 10 minutes, followed by a 1 -minute heat shock at 42°C, and then returned to ice for 3 minutes. Immediately after, 450 pL of the SOC medium was added into each tube. The cultures were combined into a 50 mL tube and incubated at 37°C with shaking at 250 rpm for 1 hour. The culture was then spread onto 20 2xYT-AG agar plates containing 100 pg / mL ampicillin and 1% glucose and incubated at 30°C for 14 hours. The diversity or the size of the generated antibody library was calcualted by counting the number of recombinant bacterial colonies that grew on the 2xYT-AG agar plates. To achieve this, 3-4 rounds of 10-fold serial dilution of the culture were performed before spreading onto the 2xYT-AG agar plates and incubated at 30°C for 14 hours. The number of colonies was counted the following day to estimate the total number of bacteria in the library. Additionally, a background control was conducted using ligated control reactions containing only the phagemid vector, to determine the number of bacteria that grew without the recombinant insert. The background count was subtracted from the total number of colonies to obtain the true size of the antibody library.

[0026] Step 8 Generation of a phage display scFv DNA library from the recombinant bacteria

[0027] Two milliliters of 2xYT broth containing 100 pg / mL ampicillin and 1% glucose (2xYT- AG) were added onto the plates with recombinant bacteria grown from Step 7. The bacterial colonies were scraped from the agar and dissolved into a suspension. The resulting suspension was then mixed with glycerol to a final concentration of 20% (v / v). This glycerol stock was either stored at -80°C for long-term storage or used as a starter culture for future use, by inoculating 500 pL of this starter culture into 250 mL of 2xYT-AG. The culture was then incubated at 37°C with shaking at 250 rpm until the optical density at 600 nm (OD600) reaches 0.5. Then, 1.75 x 1012PFU (M.O.I. of 20) of M13KO7 Helper phage (New England Biolabs) were added into the culture and incubated at 37°C with shaking at 250 rpm for another hour. Afterwards, the culture was centrifuged at 4,000 x g for 10 minutes at 30°C to separate the cells from the medium. The culture medium was discarded and replaced with 250 mL of 2xYT broth containing 100 pg / mL ampicillin and 50 pg / mL kanamycin (2xYT-AK) and incubated at 30°C with shaking at 250 rpm for 14 hours. Following incubation, the culture was centrifuged at 10,000 rpm for 30 minutes at 4°C. The phages were then precipitated from the culture supernatant by adding PEG / NaCl to the culture supernatant at a ratio of 1 :5 and incubated on ice for 1 hour. The supernatant was then centrifuged at 10,000 rpm for 30 minutes at 4°C. The precipitated phages were then resuspended in 50% (v / v) glycerol in TBS Buffer. The number of recombinant phages were quantified and stored at -20°C for future use.

[0028] Step 9 Quantification of the precipitated recombinant phage

[0029] Escherichia coli strain TGI in the log phase was prepared by first spreading on a minimal medium agar plate and incubated at 37°C for 48 hours. A single bacterial colony was then picked and inoculated into 5 mL of the 2xYT broth and incubated at 37°C with shaking at 250 rpm until the optical density at 600 nm (OD600) reaches 0.5. The culture was then aliquoted 108 pL into a 1.5 mL microcentrifuge tube. Next, 7-8 rounds of 10-folds serial dilution of the precipitated phages from the step 8 in 2xYT broth was performed, at 12 pL for each dilution. Each dilution was then added into 108 pL of the Escherichia coli TGI cells in their log phase and incubated at 37°C for 30 minutes. Subsequently, the mixture was spreaded onto 2xYT agar plates containing 100 pg / mL ampicillin and 1% glucose (2xYT-AG agar) and incubated at 37°C for 16 hours. The number of colonies was then counted to determine the titer of the recombinant phage display scFv DNA library for future use.

[0030] B. Selection of candidate recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 from the antibody library

[0031] The candidate antibodies that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica was selected from the recombinant murine single-chain variable fragment ( scFv) library by using phage display technology to obtain antibodies with specificity and high diversity against saposin-like protein 2.

[0032] The process is as follows:

[0033] Step 1 Biopanning using an immuno tube

[0034] In this step, an antigen was coated on the surface of the immuno tube. Antigen was preprared to a final concentration of 10 pg / ml in 1 mL of coating buffer solution (50 m carbonate buffer, pH 9.6) and added into the immune tube, then incubated at at 4°C for 16 hours. After incubation, the tube was washed 3 times with PBS-0.1% Tween 20. Blocking buffer (2% non-fat skim milk in PBS-0.1% Tween 20) was then added to the full volume of the tube and incubated at 25°C for 2 hours. The recombinant phage display scFv DNA library from the previous process A at a concentration of I x lO11to I x lO12PFU was prepared by mixing with 1 mL of the blocking buffer. After incubation, the blocking buffer was discarded from the immuno tube, followed by washing with PBS-0.1% Tween 20. Next, the prepared recombinant phage display scFv DNA library was added into the tube and incubated at 25°C with shaking at 200 rpm for 2 hours. Then, the immuno tube was washed 10 times with PBS-0.1% Tween 20. For the 2ndand 3rdpanning, the number of washes was increased to 20 and 30 times, respectively, to select phages that bind specifically and with high affinity. Phages that bound specifically to the immuno tube were then eluted by adding 1 mL of 100 mM triethylamine with gently shaking for 10 minutes. The eluate was then transferred into a 1.5 mL microcentrifuge tube containing 500 pL of 1 M Tris-HCl, pH 7.5, and store at 4°C for future use.

[0035] Step 2 Selection of the recombinant phage display scFv DNA library that produce antibodies in the soluble form

[0036] Seven to eight rounds of 10-folds serial dilution of the candidate phages from the step 1 was performed, at 12 pL for each dilution. Each dilution was then added into 108 pL of the Escherichia coli HB2151 cells in their log phase and incubated at 37°C for 30 minutes. Subsequently, the mixture was spreaded onto 2xYT agar plates containing 100 pg / mL ampicillin and 1% glucose (2xYT-AG agar) and incubated at 37°C for 16 hours. On the following day, a single bacterial colony was then picked and inoculated into each well of a 96-well-plate (master plate), pre-filled with 200 pL of 2xYT broth containing 100 pg / mL ampicillin and 1% glucose and incubated at 30°C with shaking at 200 rpm for 16 hours. Then 20 pL of the culture were transferred into another 96-well-plate, pre-filled with 200 pL of 2xYT broth containing 100 pg / mL ampicillin and 1% glucose (induction plate) and incubated at 30°C with shaking at 200 rpm for 2 hours. The culture was then centrifuged at 1 , 500 x g for 20 minutes at 30°C. The pellet was resuspended in the 200 pL of 2xYT broth containing 100 pg / mL ampicillin and ImM IPTG and incubated at 30°C with shaking at 200 rpm for 16 hours. On the following day, the culture was then centrifuged at 1,500 x g for 20 minutes at 4°C. Presence of antibodies in the solube form in the culture supernatant was next assessed by an enzyme-linked immunosorbent assay (ELISA).

[0037] Step 3 Detection of the antibodies in the solube form by ELISA

[0038] In this step, an antigen was coated at 0.1 pg in coating buffer solution (50 mM carbonate buffer, pH 9.6), at 100 pL per well and incubated at at 4°C for 16 hours. After incubation, the plate was washed 3 times with IxPBS. Blocking buffer (3% non-fat skim milk in IxPBS) was then added to the full volume of the well and incubated at room temperature for 1 hour. The blocking buffer was then discarded and washed 3 times with IxPBS. The culture supernatant of each clone from the step 2 was diluted with blocking buffer at a ratio of 1 : 1, added into each well at 100 pL per well and incubated at room temperature for 1 hour. The plate was washed 3 times with PBS- 0.05%(v / v) Tween20. Goat-anti E tag HRP conjugate at a concentraiton of 1 :8000 in blocking buffer was then added into each well at 100 pL per well and incubated at room temperature for 1 hour. The plate was washed 3 times with PBS-0.05%(v / v) Tween20 before adding 100 pL of TMB substrate. The plate was then incubated in the dark for 30 minutes before adding 100 pL of 0 . 1 N HC1 into each well to terminate the enzymatic reaction. The optical density (OD) of each well was measured using a microplate spectrophotometer at wavelengths of 450 nm and 630 nm to interpret the reaction results. Clones with high affinity, as defined by an OD value of at least 10 times higher than the control, were selected and further analyzed in subsequent steps.

[0039] Step 4 Nucleotide sequencing of the recombinant monoclonal antibody

[0040] In this step, the nucleotide sequence of the positive clones selected in the step 3 was analzed using DNA sequencing technique. Ten microliters were aspirated from the master plate and inoculated into 10 mL of 2xYT broth containing 100 pg / ml ampicillin and incubated at 30°C with shaking at 250 rpm for 16 hours. The culture was centrifuged at 4,000 rpm for 20 minutes at 4°C. Plasmids were extracted from the bacterial pellet using the QIAprep Spin Miniprep Kit (QIAGEN). Next, 10 microliters reaction was prepared, composing of 400 ng plasmid, 3 . 2 pmol forward or reverse primer (pCANTAB5E_Sl or pCANTAB5E_S6), lx sequencing buffer (BigDye™ Terminator v3.1), 2 pL of enzyme (BigDye™ Terminator v3.1) and a final volume adjustment to 10 pL with deionized water (RNase / DNase-free). The thermal cycler settings were as follows: 96°C for 1 minute, followed by 25 cycles of 96°C for 10 seconds, 50°C for 10 seconds and 60°C for 4 minutes, and hold at 4°C. The reaction mixture was purified with the BigDye™ XTerminator™. The nucleotide sequnces were analyzed on a 3500 / 3500xL Genetic Analyzer.

[0041] C. Assessment of antibody properties and specificity

[0042] The process is as follows:

[0043] Step 1 Antibody production and purification

[0044] Ten microliters of the master plate from the step 2 of the previous process B, were inoculated into 10 mL of 2xYT broth containing 1 00 pg / ml ampicillin and 1 % glucose and incubated at 30°C with shaking at250 rpm for 16 hours. Next, lO mL of the culture were inoculated into 1 L of 2xYT broth containing 100 pg / ml ampicillin and 1 % glucose and incubated at 30°C with shaking at 250 rpm until the optical density at wavelengths of 600 nm (ODeoo) reached approximately 0.4 on a spectrophotometer. The culture was centrifuged at 4,000 rpm for 20 minutes at 30°C. The pellet was resuspended in the 1 L of 2xYT broth containing 100 pg / mL ampicillin and IrnM IPTG and incubated at 30°C with shaking at 200 rpm for 16 hours. The culture was centrifuged at 4,000 rpm for 20 minutes at 4°C. To extract proteins from the periplasmic space using the osmotic shock technique, first the pellet was resuspended in 20 mL of lx TES buffer. Then, 33 mL of 0.2x TES buffer was added into the suspension and incubated on ice for 1 hour to allow the antibodies produced in periplasmic space to be released. The mixture was centrifuged at 4,000 rpm for 20 minutes at 4°C. The resulting supernatant was filtered through a sterile 0.45 pm syringe filter. The filtrate was stored at -20°C for future use.

[0045] Step 2 Testing of antibody specificity to antigen using sandwich ELISA technique

[0046] In this step, Fasciola gigantica antigen (FgES) was coated at 1 pg in coating buffer solution (50 rnM carbonate buffer, pH 9.6), at 100 pL per well and incubated at at 4°C for 16 hours. After incubation, the plate was washed 3 times with IxPBS. Blocking buffer (3% non-fat skim milk in IxPBS) was then added to the full volume of the well and incubated at room temperature for 1 hour. The blocking buffer was then discarded and washed 3 times with IxPBS. The recombinant monoclonal antibody against saposin-like protein 2 (scFvrFgSAP2) from the step 1 was diluted with blocking buffer at a ratio of 1 : 1, added into each well at 100 pL per well and incubated at room temperature for 1 hour. The plate was washed 3 times with PBS-0.05%(v / v) Tween20. Goat- anti E tag HRP conjugate at a concentration of 1 : 8000 in blocking buffer was then added into each well at 100 pL per well and incubated at room temperature for 1 hour. The plate was washed 3 times with PBS-0.05%(v / v) Tween20 before adding 100 pL of TMB substrate. The plate was then incubated in the dark for 30 minutes before adding 100 pL of 0. IN HC1 into each well to terminate the enzymatic reaction. The optical density (OD) of each well was measured using a microplate spectrophotometer at wavelengths of 450 nm and 630 nm to interpret the reaction results.

[0047] Step 3 Testing of antibody specificity against antigen using immunoblot technique

[0048] In this step, SDS -Polyacrylamide gel electrophoresis (SDS-PAGE) composing of 4 % stacking gel and 1 5% separating gel was first prepared. Next, the Fasciola gigantica antigen (FgES) was prepared by mixing it with the sample loading buffer to a final concentration of 1 pg / mL in a volume of 60 pL. Ten microliters of sample were loaded into each well of the stacking gel. Protein separation was performed by electrophoresis at 80 volts for 120 minutes. After separation, the proteins from the separating gel were transferred onto a nitrocellulose membrane using electroblotting at 30 volts for 16 hours. The membrane was wshed 3 times with 50 mL of IxPBS and incubated in blocking buffer (3% non-fat skim milk in IxPBS) at room temperature for 1 hour. The membrane was wshed 3 times with 50 mL of IxPBS. The recombinant monoclonal antibody against saposin-like protein 2 (scFvrFgSAP2) from the step 1 was diluted with blocking buffer at a ratio of 1 : 1, added into each well at 100 pL per well and incubated at room temperature for 2 hours, or incubated at 4°C for 16 hours. The membrane was washed 3 times with PBS- 0. l%(v / v) Tween20 . Goat-anti rabbit IgG AP conjugate at a concentration of 1 :2000 in blocking buffer was then added and incubated at room temperature for 1 hour. The membrane was washed 3 times with PBS-0.1%(v / v) Tween20 before incubating in AP buffer solution for 15 minutes. Then, NBT / BCIP substrate was added and incubated for another 15 minutes in the dark. The chemical reaction was stopped by washing the membrane with EDTA in AP buffer, and the membrane was air-dried for further analysis.

[0049] From the abovementioned process of the invention, 6 clones of the recombinant murine single-chain variable fragment ( scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica, of approximately 30 kilodaltons in molecular weight, were obtained, namely: scFvrFgSAP2 A9, scFvrFgSAP2 B6, scFvrFgSAP2 Cl, scFvrFgSAP2 E8, scFvrFgSAP2 F3 and scFvrFgSAP2 G6, with the following amino acid sequences:

[0050] 1. The recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola giganlica. clone scFvrFgSAP2 A9 has a coding sequence of the polypeptide of the antibody fragment with the amino acid sequence:

[0051] Glu Vai Leu Leu Gin Gin Ser Gly Ala Glu Leu Vai Lys Pro Gly Ala Ser Vai Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn He Gin Asp He Tyr He His Trp Vai Lys Gin Arg Pro Glu Lys Gly Leu Glu Trp He Gly Arg He Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Asn Phe Gin Asp Lys Ala Thr He Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gin Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Vai Tyr Tyr Cys Thr Arg He Pro Leu Thr Thr He He Pro Tyr Trp Tyr Phe Asp Vai Trp Gly Ala Gly Thr Thr Vai Thr Vai Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin He Vai Leu Thr Gin Ser Pro Thr Thr Met Ala Ala Ser Pro Gly Glu Lys He Thr He Thr Cys Ser Ala Ser Ser Ser He Ser Ser Asn Tyr Leu His Trp Tyr Gin Gin Lys Pro Gly Phe Ser Pro Lys Leu Leu lie Tyr Arg Thr Ser Asn Leu Ala Ser Gly Vai Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Gly Thr Met Glu Ala Glu Asp Vai Ala Thr Tyr Tyr Cys Gin Gin Gly Ser Ser He Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu He Lys

[0052] 2. The recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola giganlica. clone scFvrFgSAP2 B6 has a coding sequence of the polypeptide of the antibody fragment with the amino acid sequence:

[0053] Gin Vai Gin Met Gin Gin Ser Gly Pro Gin Leu Vai Arg Pro Gly Ala Ser Vai Lys lie Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met His Trp Vai Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He Gly Met lie Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gin Lys Phe Lys Asp Lys Ala Thr Leu Thr Vai Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Vai Tyr Tyr Cys Ala Arg Ser Arg Gly Gly Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Vai Leu Met Thr Gin Thr Pro Leu Ser Leu Pro Vai Ser Leu Gly Asp Gin Ala Ser He Ser Cys Arg Ser Ser Gin Ser Leu Vai His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr Lys Vai Ser Asn Arg Phe Ser Gly Vai Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He Ser Arg Vai Glu Ala Glu Asp Leu Gly Vai Tyr Phe Cys Ser Gin Ser Thr His Vai Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 3. The recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica. clone scFvrFgSAP2 Cl has a coding sequence of the polypeptide of the antibody fragment with the amino acid sequence:

[0054] Gin Vai His Leu Gin Gin Ser Gly Ser Glu Leu Arg Ser Pro Gly Ser Ser Vai Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Vai Phe Pro He Ala Tyr Met Ser Trp Vai Arg Gin Lys Pro Gly His Gly Phe Glu Trp He Gly His He Leu Pro Ser He Gly Arg Thr He Tyr Gly Glu Lys Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Vai Ser Asn Thr Ala Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala lie Tyr Tyr Cys Ala Arg Gly Gly Vai Trp Arg Tyr Asp Vai Gly Phe Ala Tyr Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Vai Met Thr Gin Ser Pro Ser Ser Leu Ala Vai Ser Vai Gly Glu Lys Vai Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Tyr Ser Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Vai Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Vai Lys Ala Glu Asp Leu Ala Vai Tyr Tyr Cys Gin Gin Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[0055] 4. The recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola giganlica. clone scFvrFgSAP2 E8 has a coding sequence of the polypeptide of the antibody fragment with the amino acid sequence:

[0056] Glu Vai Gin Leu Vai Glu Ser Gly Gly Asp Leu Vai Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Thr Thr Ser Gly Phe Thr Phe Arg Ser Tyr Ala Met Ser Trp Vai Arg Gin Thr Pro Asp Lys Arg Leu Glu Trp Vai Ala Thr He Ser Ser Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Vai Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu Gin Vai Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Asp Tyr Asp Gly Phe Pro Ala Trp Phe Ala Phe Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Vai Thr He Ser Cys Arg Ala Ser Gin Asp He Ser Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Vai Lys Leu Leu lie Tyr Tyr Thr Ser Arg Leu His Ser Gly Vai Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr He Ser Asn Leu Glu Gin Glu Asp He Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys

[0057] 5. The recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola giganlica. clone scFvrFgSAP2 F3 has a coding sequence of the polypeptide of the antibody fragment with the amino acid sequence: Gin Vai Gin Leu Lys Gin Ser Gly Pro Glu Leu Vai Arg Pro Gly Ala Ser Vai Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Vai Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He Gly Met He Asp Pro Ser Asp Ser Glu Thr Thr Leu Asn Gin Lys Phe Lys Asp Lys Ala Thr Leu Thr Vai Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Vai Tyr Tyr Cys Ala Arg Ser Arg Gly Gly Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Vai Vai Met Thr Gin Thr Pro Leu Ser Leu Pro Vai Ser Leu Gly Asp Gin Ala Ser He Ser Cys Arg Ser Ser Gin Ser Leu Vai His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr Lys Vai Ser Asn Arg Phe Ser Gly Vai Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He Ser Arg Vai Glu Ala Glu Asp Leu Gly Vai Tyr Phe Cys Ser Gin Ser Thr His Vai Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

[0058] 6. The recombinant murine single-chain variable fragment (scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola giganlica. clone scFvrFgSAP2 G6 has a coding sequence of the polypeptide of the antibody fragment with the amino acid sequence:

[0059] Glu Vai Gin Leu Leu Glu Ser Gly Gly Asp Leu Vai Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Thr Thr Ser Gly Phe Thr Phe Arg Ser Tyr Ala Met Ser Trp Vai Arg Gin Thr Pro Asp Lys Arg Leu Glu Trp Vai Ala Thr He Ser Ser Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Vai Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu Gin Vai Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Asp Tyr Asp Gly Phe Pro Ala Trp Phe Ala Phe Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Vai Thr He Ser Cys Arg Ala Ser Gin Asp He Ser Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Vai Lys Leu Leu He Tyr Tyr Thr Ser Arg Leu His Ser Gly Vai Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Ser Leu Thr He Ser Asn Leu Glu Gin Glu Asp He Ala Thr Tyr Phe Cys Gin Gin Gly His Thr Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys

[0060] Best Mode or Preferred Embodiment of the Invention

[0061] Best mode or preferred embodiment of the invention is as provided in the description of the invention.

Claims

Claims1 . A recombinant murine single-chain variable fragment ( scFv) that specifically binds to saposin-like protein 2 of the liver fluke Fasciola gigantica. comprising 6 clones, namely: scFvrFgSAP2 A9, scFvrFgSAP2 B6, scFvrFgSAP2 Cl, scFvrFgSAP2 E8, scFvrFgSAP2 F3 and scFvrFgSAP2 G6.

2. The recombinant murine single-chain variable fragment (scFv) that specifically bind to saposin-like protein 2 of the liver fluke Fasciola gigantica according to claim 1, wherein the clone scFvrFgSAP2 A9 has a coding sequence of the polypeptide of the antibody fragment:Glu Vai Leu Leu Gin Gin Ser Gly Ala Glu Leu Vai Lys Pro Gly Ala Ser Vai Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn He Gin Asp He Tyr He His Trp Vai Lys Gin Arg Pro Glu Lys Gly Leu Glu Trp He Gly Arg He Asp Pro Glu Asn Gly Asn Thr Lys Tyr Asp Pro Asn Phe Gin Asp Lys Ala Thr He Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gin Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Vai Tyr Tyr Cys Thr Arg He Pro Leu Thr Thr He He Pro Tyr Trp Tyr Phe Asp Vai Trp Gly Ala Gly Thr Thr Vai Thr Vai Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin He Vai Leu Thr Gin Ser Pro Thr Thr Met Ala Ala Ser Pro Gly Glu Lys He Thr He Thr Cys Ser Ala Ser Ser Ser He Ser Ser Asn Tyr Leu His Trp Tyr Gin Gin Lys Pro Gly Phe Ser Pro Lys Leu Leu lie Tyr Arg Thr Ser Asn Leu Ala Ser Gly Vai Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Gly Thr Met Glu Ala Glu Asp Vai Ala Thr Tyr Tyr Cys Gin Gin Gly Ser Ser He Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu He Lys.

3. The recombinant murine single-chain variable fragment (scFv) that specifically bind to saposin-like protein 2 of the liver fluke Fasciola gigantica according to claim 1 , wherein scFvrFgSAP2 B6 has a coding sequence of the polypeptide of the antibody fragment:Gin Vai Gin Met Gin Gin Ser Gly Pro Gin Leu Vai Arg Pro Gly Ala Ser Vai Lys He Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met His Trp Vai Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He Gly Met lie Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gin Lys Phe Lys Asp Lys Ala Thr Leu Thr Vai Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Vai Tyr Tyr Cys Ala Arg Ser Arg Gly Gly Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Vai Leu Met Thr Gin Thr Pro Leu Ser Leu Pro Vai Ser Leu Gly Asp Gin Ala Ser He Ser Cys Arg Ser Ser Gin Ser Leu Vai His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr Lys Vai Ser Asn Arg Phe Ser Gly Vai Pro Asp Arg Phe Ser Gly SerGly Ser Gly Thr Asp Phe Thr Leu Lys He Ser Arg Vai Glu Ala Glu Asp Leu Gly Vai Tyr Phe Cys Ser Gin Ser Thr His Vai Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys.

4. The recombinant murine single-chain variable fragment (scFv) that specifically bind to saposin-like protein 2 of the liver fluke Fasciola gigantica according to claim 1 , wherein scFvrFgSAP2 Cl has a coding sequence of the polypeptide of the antibody fragment:Gin Vai His Leu Gin Gin Ser Gly Ser Glu Leu Arg Ser Pro Gly Ser Ser Vai Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Vai Phe Pro He Ala Tyr Met Ser Trp Vai Arg Gin Lys Pro Gly His Gly Phe Glu Trp He Gly His He Leu Pro Ser He Gly Arg Thr He Tyr Gly Glu Lys Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Vai Ser Asn Thr Ala Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala lie Tyr Tyr Cys Ala Arg Gly Gly Vai Trp Arg Tyr Asp Vai Gly Phe Ala Tyr Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Vai Met Thr Gin Ser Pro Ser Ser Leu Ala Vai Ser Vai Gly Glu Lys Vai Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Tyr Ser Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Vai Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Vai Lys Ala Glu Asp Leu Ala Vai Tyr Tyr Cys Gin Gin Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys.

5. The recombinant murine single-chain variable fragment (scFv) that specifically bind to saposin-like protein 2 of the liver fluke Fasciola gigantica according to claim 1 , wherein scFvrFgSAP2 E8 has a coding sequence of the polypeptide of the antibody fragment:Glu Vai Gin Leu Vai Glu Ser Gly Gly Asp Leu Vai Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Thr Thr Ser Gly Phe Thr Phe Arg Ser Tyr Ala Met Ser Trp Vai Arg Gin Thr Pro Asp Lys Arg Leu Glu Trp Vai Ala Thr He Ser Ser Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Vai Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu Gin Vai Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Asp Tyr Asp Gly Phe Pro Ala Trp Phe Ala Phe Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Vai Thr He Ser Cys Arg Ala Ser Gin Asp He Ser Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Vai Lys Leu Leu lie Tyr Tyr Thr Ser Arg Leu His Ser Gly Vai Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr He Ser Asn Leu Glu Gin Glu Asp He Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys.

6. The recombinant murine single-chain variable fragment (scFv) that specifically bind to saposin-like protein 2 of the liver fluke Fasciola gigantica according to claim 1 , wherein scFvrFgSAP2 F3 has a coding sequence of the polypeptide of the antibody fragment:Gin Vai Gin Leu Lys Gin Ser Gly Pro Glu Leu Vai Arg Pro Gly Ala Ser Vai Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Vai Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He Gly Met He Asp Pro Ser Asp Ser Glu Thr Thr Leu Asn Gin Lys Phe Lys Asp Lys Ala Thr Leu Thr Vai Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Vai Tyr Tyr Cys Ala Arg Ser Arg Gly Gly Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Vai Vai Met Thr Gin Thr Pro Leu Ser Leu Pro Vai Ser Leu Gly Asp Gin Ala Ser He Ser Cys Arg Ser Ser Gin Ser Leu Vai His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr Lys Vai Ser Asn Arg Phe Ser Gly Vai Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He Ser Arg Vai Glu Ala Glu Asp Leu Gly Vai Tyr Phe Cys Ser Gin Ser Thr His Vai Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys.

7. The recombinant murine single-chain variable fragment (scFv) that specifically bind to saposin-like protein 2 of the liver fluke Fasciola gigantica according to claim 1 , wherein scFvrFgSAP2 G6 has a coding sequence of the polypeptide of the antibody fragment:Glu Vai Gin Leu Leu Glu Ser Gly Gly Asp Leu Vai Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Thr Thr Ser Gly Phe Thr Phe Arg Ser Tyr Ala Met Ser Trp Vai Arg Gin Thr Pro Asp Lys Arg Leu Glu Trp Vai Ala Thr He Ser Ser Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Vai Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr Leu Gin Vai Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Asp Tyr Asp Gly Phe Pro Ala Trp Phe Ala Phe Trp Gly Gin Gly Thr Leu Vai Thr Vai Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Vai Thr He Ser Cys Arg Ala Ser Gin Asp He Ser Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Vai Lys Leu Leu He Tyr Tyr Thr Ser Arg Leu His Ser Gly Vai Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Ser Leu Thr He Ser Asn Leu Glu Gin Glu Asp He Ala Thr Tyr Phe Cys Gin Gin Gly His Thr Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys.