Cloning, Expression Analysis, and Detection of the Vitellogenin in the Chinese Black Sleeper Bostrychus sinensis

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Introduction
In recent years, environmental estrogens (EEs) as an endocrine disrupting chemical (EDC) have attracted extensive concern [1] and were divided into two categories including natural and synthetic estrogens [2]. Studies have shown that fsh exposed to estrogenic chemicals may lead to populationrelated efects [3], including reduced production of viable ofspring and male sexual reversal [4]. EEs can be transmitted to organisms through inhalation, food intake, and direct contact [5]. Due to its high lipophilic nature, it accumulates in adipose tissue and has a long half-life [6], making the pollution phenomenon to deteriorate [7]. Vitellogenin (Vg), a precursor protein, has become a common biomarker used to determine the level of estrogen or estrogen analogue contamination in oviparous animals [8] and is produced by the liver of oviparous vertebrates in response to estrogen [9]. Te Vg gene is silenced in male and juvenile fsh [10], while it is activated in the stimulation of exogenous estrogen compounds [11]. Te Vg synthesized in male fsh is delivered into the blood and has kept at a high level because of no ovaries [12]. Terefore, the abnormal elevation of Vg in male fsh can indirectly indicate the pollution status of estrogens in the aquatic environment [13]. In previous studies, the natural estrogen (E2, Estradiol) was the preferred hormone for inducing fsh feminization, which has been demonstrated in previous research [14]. E2 is highly potent with high environmental persistence and a tendency to bioconcentrate in organisms [15] and can disrupt reproductive processes in fsh at low concentrations that occur in the aquatic environment [16].
At present, most of the studies about the cloning and expression of the Vg gene in fsh focus on freshwater [17,18]. However, freshwater is diferent from marine environments due to diverse physiological structure of fsh living to some extent. Terefore, it is more accurate to monitor the changes of the marine environment based on the marine fsh.
Te Chinese Black Sleeper, Bostrichthys sinensis, belonging to Periforms, Eleotridae, Bostrichthys [19], is a warm-water euryhaline fsh distributed in the southeast coast of China and Taiwan Strait of China [20], which is sensitive to estrogen pollutants. Because of its strong settlement [21], it can refect the pollution situation along the estuarine in a certain region for a long time. According to the long-term detection of the research found that B. sinensis is sensitive to environmental changes, especially estrogen [22]. Low concentrations of estrogen can cause changes in the gonads or related physiological and biochemical indicators [23].
Terefore, to obtain a more efective method for detecting EE contamination, the changes in relative expression under diferent E2 concentrations were studied to enhance the healthy development of the mariculture and marine environment.

Fish Husbandry.
B. sinensis is kept in the laboratory. E2 was added to the frst, second, and third groups of male fsh with estradiol concentrations of the low-dose group (10 μg/ L), medium-dose group (50 μg/L), and high-dose group (100 μg/L), respectively. Te fourth and ffth groups were males and females without estradiol as controls, and all groups had 12 fsh.
Samples were divided into fve treatment groups with each group reared in a 30 cm × 20 cm×15 cm glass container with 3 L fltrated seawater. Te values of temperature and salinity were 25°C and 5‰ to 10‰, respectively. Adjust the biological rhythm to day: night � 10 : 14.

2.2.
Acquisition of the Full-Length cDNA of BsVg. Total RNA was extracted from the liver tissue of the male B. sinensis induced by E2 solution and was reversely transcribed into cDNA based on the protocol 5× All-In-One MasterMix (with AccuRT Genomic DNA Removal Kit).
We have downloaded 16 fsh gene sequences from the NCBI database and designed PCR primers accordingly. Te primers are Vg-CDS-F: ATGAGAGYNGTTGTRCTWGC// Vg-CDS-R: CAGCCTTTCCACAAGWCCAC. Te Vg cDNA was amplifed, and PCR products were conducted through agarose gel electrophoresis. Te cDNA fragment was cloned into T4 vector and transformed into E. coli [24]. According to the results of the blue-white spot screening, a few colonies were selected for amplifcation. Ten, the plasmid was extracted and sequenced by Xiamen Bosheng Biotechnology Company.
Te full-length cDNA sequence of vitellogenin expression was further determined by designing RACE PCR primers at both ends. Te designed primer sequence is Vg51 ( During the step, Vg31 + XP1378 was extended for 2 min. Vg51 + XP1379 was extended for 1.5 min. At a temperature of 60°C, each cycle was expanded for 25 cycles. PCR was repeated with 1 μL of each product of the abovementioned process, in which the extension time of Vg32 + XP1378 and Vg52 + XP1379 was 1.5 min, and the cycle time was 20 times at 60°C. Te fnal product was subjected to agarose gel electrophoresis and sequencing. Te abovementioned two sequencing results were spliced to obtain the preliminary sequence (Annex 1), and the sequencing results were compared (Annex 2).

Cloning, Construction, and Protein Induction of Recombinant Plasmid.
For expression of Vg, two primers were designed for cloning in Table 1 and the reaction procedure of the PCR experiment is shown in Table 2. Te Vg PCR fragment of 825 bp was amplifed and cloned into the T4 vector, and positive clone was confrmed by sequencing. Te 825 bp fragment was released with SalI/ BamHI from T4 vector and inserted into pMAL-c5x plasmid that was digested with SalI/BamHI.
Te recombinant pMAL-Vg-c5x plasmid was transformed into an E. coli BL21 (DE3) bacterial strain [24]. Te strain was subjected to thermal shock at 42°C for 45 s and was placed on ice for 2 min and then coated with a plate (100 μg/mL ampicillin). After incubation at 37°C overnight, suitable colonies were selected. Four induction conditions of 16°C, 20°C, 28°C, and 37°C were applied, and the optimal efect was compared. Finally, 16°C was determined as the best condition for the following large amount of induction and conduct the mass induction.
Samples collected by centrifugation were placed in the Bioruptor ultrasonic crushing apparatus. Te crushing conditions were as follows: continuous 15 s at 4°C and suspension of 15 s for a total length of 2 min for 20 consecutive cycles. Te supernatant and precipitation were separated by centrifugation for 20 min at a speed of 1006.2 x g at 4°C. Te protein concentration was measured by using the Qubit ™ Protein Assay Kit, and the proteins were further identifed by SDS-PAGE electrophoresis.

Immunohistochemistry and Histological Examination
Procedure. Te purifed protein (Vg) was transferred to Xiamen Bo sheng Biological Company for monoclonal antibody preparation. Following the SABC kit instructions (SA1020) to detect the expression site of Vg in sexually mature female fsh, the prepared monoclonal antibody above was used for immunohistochemistry.
We fx the intestine, muscle, fshtail, hepatopancreas, ovary, and pronephric kidney in 4% paraformaldehyde. Te main procedures included tissue dehydration, embedding, sectioning, and HE staining, and the tissue dehydration is shown in Table 3.
After embedding, parafn sections were made. Te section thickness was set to 5 μm∼10 μm for sectioning, and the thickness was adjusted according to the sectioning efect. Te slices were rinsed in 30% ethanol solution for a while and spread in warm water at 24°C. After the slices were removed, they were baked in a constant temperature oven at 60°C for 2 h and then dewaxed and rehydrated, as shown in Table 4.
Te dewaxed slices were rinsed with running water for HE staining. Parafn sections dyed completely by HE must be thoroughly dehydrated and transparent before sealing with neutral resin. Te HE dyeing procedure is shown in Table 5.
IHC experiments were performed using the pressure cooker method and the antigen repair method, referring to the SABC kit instructions (SA1020).
In the IHC experiment, the powder form of antigen repair solution was dissolved in distilled water and heated to boiling in an autoclaved pot. Te sections were immersed in the antigen repair solution and soaked in an autoclaved pot at 121°C for 5 min. Te sections were treated with 1‰ TritonX-100 for 10 min, and the sections were washed with 0.01 mol/L PBS three times for 3 min each on a shaker. Te sections were incubated with 3% hydrogen peroxide for 10 min at room temperature and washed with 0.01 mol/L PBS three times for 3 min each on a shaker, and the blank control group and experimental group of each section were marked clearly with a histochemical pen.
Te samples were incubated in a 37°C constant temperature incubator for 30 min with a drop of 5% BSA blocking solution and then dried. Te samples were     Biotin-labeled goat antimouse IgG (1 : 100) was dropped and incubated at 37°C for 30 min, and then it was washed three times with PBS for 3 min each. SABC (1 : 100) was added drop, incubated at 37°C for 30 min, and washed with PBS three times for 3 min each. During color development, DAB reagent components A and B in the DAB horseradish peroxidase kit were added to each section with a drop of 100 μL at 1 : 1, and the reaction was performed in a 37°C incubator for 5 min (yellow color was considered as positive reaction). After completing the above operation, the DAB liquid on the glass slide was washed of with running water and 100 μL hematoxylin dyeing solution was added for 30 s. After color development, the residual liquid on the glass slide was washed of with running water. Drop the sealer (glycerin: distilled water/PBS � 9 : 1) on one side of the glass slide and gently place the cover slip to avoid bubbles.

Exposure Experiment.
During the feeding process, the water was changed every 2 days. On the 3rd, 8th, 11th, and 14th days, three fsh were randomly captured in one group to obtain the liver and RNA was extracted and transcribed into cDNA for qRT-PCR. Te relationship between E2 diferent concentrations at diferent times and Vg expression of B. sinensis was studied. Te qRT-PCR assays were referred to the BlasTaq ™ 2X qPCR MasterMix kit, and the procedures are shown in Table 6.

Te
Full-Length cDNA of BsVg. We download 16 fsh gene sequences with close genetic relationships from the NCBI database and design PCR primers by DNAMAN for comparison. Te cDNA PCR product from the female B. sinensis liver was conducted agarose gel electrophoresis using the primers mentioned above. Ten, the cDNA sequence of Vg was further prepared by RACE PCR (Figure 1(b)). According to the size of the Vg, the molecular weight was determined to be about 4600−5000 bp (Figure 1(a)), so the strip was recycled and sent for sequencing. Te full-length cDNA of BsVg and its encoding amino acid sequence are shown in Annex 1, and agarose gel electrophoresis is shown in Figure 1. Te homology of the amino acid sequences of BsVg is shown in Annex 2.
We obtain the full-length cDNA of BsVg, which flls the blank of the Vg gene bank in B. sinensis. Te BLAST comparison with other similar fsh shows that the similarity was between 47.28% and 65.28%, and the specifcity was maintained at more than 30%.

Expression and Purifcation Recombinant Plasmid.
Te soluble protein was obtained by prokaryotic expression of the fusion protein based on the cloned fragment (825 bp) from the full-length cDNA of BsVg (4738 bp).
Te Vg PCR fragment of 825 bp was amplifed and cloned into T4 vector and was released with SalI/BamHI from T4 vector and inserted into pMAL-c5x plasmid, which was digested with SalI/BamHI (Figure 2).
Te recombinant plasmid was transferred to E. coli BL21 for Vg expression. Efcient fusion expression was obtained by IPTG (0.05 mM) at an induced temperature of 16°C, and the expressed target protein (680 amino acids, 75 kDa) existed in a soluble state, accounting for more than 25% of the total soluble protein. SDS-PAGE analysis of the induced expression protein at diferent temperatures is shown in Figure 3. Te maltose binding protein (MBP) fusion expression system and the selected 16°C induction conditions determined that the fusion protein was highly expressed and easy to purify by using the afnity chromatography column.
Te highly expressed protein obtained at 16°C could be detected in the supernatant with fewer inclusion bodies. It is in line with our expectations and provides a good foundation for future experiments.
Te recombinant Vg fusion protein of B. sinensis was further purifed by a maltose-binding protein-tag purifcation column (Dextrin Beads 6FF). Te purifed product was identifed by SDS-PAGE ( Figure 4).
Enzymatic digestion of fusion protein monomers with Factor Xa was performed at 20°C, and the time was 28, 32, and 36 h. Fusion protein and Factor Xa are mixed at 20 : 1 (concentration ratio). Te results of SDS-PAGE analysis showed that the fusion protein without digestion almost had no target protein Vg at a molecular weight of 35 kDa, while the target protein appeared at 35 kDa after digestion and increased with the extension of digestion time (Figure 5). Te obtained protein concentration (1.25 mg/mL) could be accurately measured by using the Qubit ™ Protein Assay Kit.

Immunohistochemical (IHC) Tissue Section Detection.
Monoclonal antibodies and purifed Vg were identifed by Western blot in Figure 6. Following the SABC kit instructions to detection, the expression site of Vg in sexually mature female fsh using the monoclonal antibody was prepared by Xiamen Bo sheng Biological Company. We detected Vg expression in the fshtail, hepatopancreas, intestine, muscle, ovary, and pronephric kidney. Te results of hematoxylin-eosin staining are shown in Figure 7, and the morphology of the entire cell tissue is clearly visible.  Immunohistochemical tissue sections show that the monoclonal antibody prepared has certain specifcity and can be well applied to tissue detection in Figure 8.
According to the staining results, the expression sites of Vg in sexually mature female fsh are mainly distributed in the fshtail, hepatopancreas, intestine, muscle, ovary, and pronephric kidney. Te tail has an obvious dyeing efect, and the main guess is that the tail has abundant blood vessels.

qRT-PCR Analysis.
Based on the full-length cDNA of BsVg, qRT-PCR assays were used to analyze the relative expression of Vg with E2 induced.
Te low-dose group (10 μg/L) was found to increase on day 3, peak on day 8, and then decrease gradually. Te medium-dose group (50 μg/L) rose to the highest level on day 8 and then gradually declined, but its level was the highest on day 14 in all groups. Te high-dose group (100 μg/ L) had a low level on day 3, a high level on day 8 and day 11, and a signifcant decrease on day 14 ( Figure 9).
Analysis of Vg expression in diferent concentrations of E2 and diferent exposure time by qRT-PCR indicated that Vg was not or low expressed in normal male fsh, but it was expressed in female fsh. However, the expression level of male fsh began to increase with time, there was an increase in E2 concentration, and the expression level was higher than that of Vg in female fsh. It was proved that the Vg and its expression in male B. sinensis could accurately refect prolonged pollution status and the degree of environmental estrogens.

Discussion
At present, in the process of life science research and the production of biological products [8], the preparation of recombinant proteins using expression vectors is one of the most important technologies [25]. Te construction of an efective expression vector is the basic requirement for the expression of target genes [24], and it is also an important factor afecting the gene expression level and protein activity [14].
In addition, B. sinensis is a commercially important fsh in southeastern China and Taiwan Strait of China [26] for its   Journal of Applied Ichthyology delicious taste, rich nutrition, and a believed traditional medical function of promoting wound healing. Nowadays, this species is gradually becoming an important mariculture fsh in the coastal areas of Zhejiang, Fujian, and Guangdong provinces, China [22]. However, such as other farming fsh species in China [27], B. sinensis has sufered from many kinds of estrogen contamination. Terefore, better understanding the mechanisms underlying the Vg response of B. sinensis to EEs will help us develop strategies for the management of the environment and enhance the healthy development of the mariculture of this commercial fsh species. In our research, total RNA was extracted from the liver tissue of male B. sinensis, and the full-length cDNA of BsVg was obtained by reverse transcription amplifcation. Te Vg fusion protein with MBP-tag was formed by the construction of a recombinant plasmid, and the optimal induced expression condition (16°C, 0.05 mM IPTG) was found. Te highly expressed protein obtained at 16°C could be detected in the supernatant and precipitation after centrifugation, mainly in the supernatant. It provides a good foundation for our future experiments.
In this experiment, the obtained Vg was transferred to the Xiamen Bosheng Biological Company for preparation of monoclonal antibodies. Te expression of Vg in sexually mature female fsh was mapped by the immunohistochemistry assay. Te results show that Vg mainly distributed in the fshtail, hepatopancreas, intestine, muscle, ovary, pronephric kidney, and the tail is most obvious.
Based on the full-length cDNA of BsVg, the relative expression of Vg induced by E2 was analyzed by the qRT-PCR method. Tree groups of diferent concentrations of E2 exposure were designed in 3, 8, 11, and 14 days from the B. sinensis liver. Results show that the Vg expression and exposure concentration have an obvious dose efect relationship, and the most obvious expression is on the eighth day of the exposure cycle.
Terefore, the regulation and expression of Vg could better refect the pollution status of environmental estrogen, and it is more accurate and practical for environmental monitoring and evaluation as a biomarker of environmental estrogen [10].
Our study shows that Vg could be used as a biological monitoring index in marine environments, and the fulllength cDNA of BsVg was obtained. Meanwhile, localization and quantitative detection methods of Vg expression were also achieved. Te subsequent experiments of development in colloidal gold strips after this research would be established to provide a highly efcient and convenient environmental pollution detection method [28], and the colloidal gold strip depends on the monoclonal antibody prepared in this paper. At present, the detection range and type of the strip are still under further testing, and we will continue to carry out relevant research in this respect.

Conclusion
Te full-length cDNA cloning of the BsVg gene and the expression and purifcation of the corresponding fusion protein of this gene through recombinant plasmid construction were realized in this study. Te prepared antiVg monoclonal antibody of B. sinensis enables the localization detection of Vg in its tissues. Te expression level of the Vg gene can efectively refect the estrogenic efects of environmental estrogens on B. sinensis. Te experimental results of this study provide a solid scientifc basis for the application of B. sinensis and their Vg in the monitoring of aquatic environmental estrogen pollution, and the prepared monoclonal antibody can be used for the research of the colloidal gold detection method subsequently.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
Tese animal experiments were approved by the Institutional Animal Care and Use Committee and were in strict accordance with good animal practice as defned by the Xiamen University Laboratory Animal Center (XMULAC20200022).

Conflicts of Interest
Te authors declare that they have no conficts of interest.