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Plasmid Based Expression and Bioactivity Evaluation of Caprine Growth

Hormone Gene Cloned from a Local Pakistani Goat Breed, Beetal

Article in International Journal of Agriculture and Biology · January 2014

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
13–1105/2014/16–3–634–638
http://www.fspublishers.org

Full Length Article

Plasmid Based Expression and Bioactivity Evaluation of Caprine Growth

Hormone Gene Cloned from a Local Pakistani Goat Breed, Beetal
Hamama Islam Butt1, Mirza Imran Shahzad2, Qudsia Bashir3, Muhammad Arif Nadeem Saqib4 and Azra Khanum5,6*
1
Department of Biochemistry, University of Gujrat, Gujrat, Pakistan
2
Department of Biochemistry, University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur,
Bahawalpur, Pakistan
3
Atta ur Rehman School of Applied Biosciences, National University of Science and Technology, Islamabad, Pakistan
4
Pakistan Medical Research Council, Islamabad, Pakistan
5
Department of Biochemistry, PMAS, Arid Agriculture University Rawalpindi, Murree Road, Rawalpindi - 46300, Pakistan
6
Institute of Natural and Management Sciences Rawalpindi, 31-D Satellite Town Rawalpindi - 46300, Pakistan
*For correspondence: [email protected]

Abstract

Growth hormone cDNA of Beetal goat (Capra hircus), an indigenous breed of Punjab, Pakistan was amplified by RT PCR
and gene including leader sequence was cloned in pTZR57 cloning vector. The cGH-pTZR57 clone was confirmed by
restriction digestion and sequence analyses before finally sub-cloning the gene in pND - a mammalian expression vector. The
clones were again confirmed by restriction digestion and PCR analyses. Highly purified, super coiled recombinant caprine
growth hormone-pND (rcGH-pND) construct was used to transfect Vero cell lines for expression studies. The in vitro
expression of cGH was determined by dot-ELISA. After confirming its in vitro cell line based expression, rcGH-pN, pND
constructs and phosphate buffer saline (PBS) were separately injected to 4 weeks old balb/c female mice intramuscularly.
Total forty five animals were used to determine the biological activity and randomly divided in to three groups with fifteen
mice in each group. Five animals from each group were used to monitor the in vivo biological activity by evaluating the body
weight gain and tibia epiphyseal width assays from zero week up to four weeks with one week gap. Significant increase
(P<0.05) gain in body weight and in tibia epiphyseal width was observed in animals inoculated with rcGH-pN. Thus it is
concluded that recombinant plasmid of cGH cDNA may be used as supplement to increase the meat production. The effects of
the same plasmid on milk production may be checked in future experiments using Beetal goat. © 2014 Friends Science
Publishers

Keywords: Caprine growth hormone; Mammalian expression vector pND; Vero cell line; Beetal growth hormone

Introduction glycosylated protein hormone secreted from the anterior

pituitary gland having 190 amino acids. Its biological effects
The economy of Pakistan is mainly dependent upon are broadly classified as either somatogenic or metabolic
agriculture and livestock is the second important sector of which results in linear growth and milk production of the
agriculture. Pakistan is rich in its livestock, however, due to ruminant system (Pell and Bates, 1987). The somatogenic
urbanization and increasing population size, demands for effects are mediated by insulin like growth factor-1 (IGF-1)
meat and milk is also increasing. Since past, efforts are being while the metabolic effect involves a variety of tissues and
made to cope with these demands by different conventional general metabolism of carbohydrate, lipid, protein, and
methods such as selection breeding, improved management, minerals essential for growth (Etherton and Bauman, 1998).
establishment of more dairy herds and utilization of various For different biological roles and commercial application in
scientific techniques (Zhang et al., 2013). One of these animal husbandry, GH has been cloned from a number of
methods is to strength existing and/or create new species and expressed in various expression systems like
infrastructure to facilitate the milk supply along with the prokaryotic (Wingfield et al., 1987; Mukhopadhyay and
stress on milk production (Chattha et al., 2013). Beside all Sahni, 2002; Khan et al., 2007, Khalid et al., 2008) and
these conventional methods, one of the latest scientific eukaryotic system (Hawkins and Nakamura, 1999) to
techniques is the use of recombinant technology for the produce high levels of recombinant protein, which is being
production of valuable proteins like bovine growth hormone commercially used for enhancement of milk and meat
(bGH). It is a multifunctional naturally occurring non- production in cattle (Baldi, 1999; Bauman, 1999).

To cite this paper: Butt, H.I., M.I. Shahzad, Q. Bashir, M.A.N. Saqib and A. Khanum, 2014. Plasmid based expression and bioactivity evaluation of caprine
growth hormone gene cloned from a local Pakistani goat breed, Beetal. Int. J. Agric. Biol., 16: 634‒638
 Plasmid Based Expression and Bioactivity Evaluation of cGH / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

Because of high genetic potentials and low DH5α cells was also refreshed by streaking and growing on
productivity of milk and meat in most of South East Asian Lauria-Bertani (LB) media prepared by the method
cattle and small ruminant breeds, the exogenous mentioned by Maniatis et al. (1989). Plasmid DNA of pND
administration of recombinant homologous GH approaches vector was extracted to be used in sub-cloning experiment.
on one hand provide the key for boosting the productivity of This vector contains constitutive immediate early promoter
ruminants to increase the food output (meat or milk) per unit and enhancer sequences from cytomegalovirus (CMV) and
of food source input. This is because of reduction in animal 1000 bp intron sequence for optimal gene expression under
waste products and expenditures for animal feed production in vitro and in vivo conditions. Before starting experiments,
(Bauman, 1992). On the other hand, the state of the art the protocols were reviewed and approved by the ethical
plasmid based expression technology is getting popular in committee of PMAS Arid Agriculture University
recent years (Patil et al., 2005). For example, to screen the Rawalpindi.
targets identified from genomic projects, to shuffle
molecules for vaccination or direct in vivo production of Sub-cloning of cGH in pND
hormones and other therapeutics or preventive applications
(Draghia-Akli et al., 2006). This economically feasible gene The cGH-pTZR57 construct was restricted with Xba I and
delivery technology and plasmid mediated gene transfer is Bam HI to produce the 692 bp long cGH gene with leader
also emerging as an excellent candidate for agriculture sequence. Similarly pND vector was modified by restriction
applications to optimize production and animal welfare digestion with Nhe I and Bam HI enzymes. The fragments
(Draghia-Akli and Fiorotto, 2004; Cunningham, 2005). were purified from gel by gel extraction kit (Fermentas,
Goats (Capra hircus) are fastest growing ruminants in USA) and purified products were ligated with T4 ligase
Pakistan and about 25 goat breeds are in the country and using standard ligation procedure provided by Supplier
two wild relative such as Markhor and Ibex (Khan et al., (Roche, USA). The ligation mixture was then transformed
2008; Shahzad et al., 2012). These are important for poor into chemically competent B10 strain of E. coli. The
livestock farmers to make a living and according to recent positive clones were confirmed by restriction digestion
data over 64 million goats are available in Pakistan (GOP, using EcoRV and agarose gel electrophoresis.
2013). Some species are good for meat or milk production. Expression Studies
For example, Beetal goats are famous for its milk and meat
production and is called poor man’s cow (Iqbal et al., 2008; DNA used for expression studies was prepared by Quantum
Qudus et al., 2013). These goats are found in almost all prep™ Plasmid Midiprep kit (Bio-Rad, USA). DNA
irrigated areas of the Punjab including districts of Jhelum, concentration was determined by spectrophotometer at
Gujarat, Mandi-Bahaudddin, Sialkot, Gujranwala, Lahore, optical density of 260 nm and 280 nm. The ratio of 260/280
Sheikhupura, Faisalabad, Sargodha, Jhang, Multan, Sahiwal nm was used to determine the quality of DNA. Most of
and Okara (www.haasil-foundation.com/). DNA samples gave ratio between 1.7 and 2.0. The samples
Keeping in view the importance of Beetal breed’s with lower ratio were retreated with chloroform-
contribution towards meat and milk production, this study isoamylalcohol to remove proteins. The revived Vero cells
was designed to clone the caprine growth hormone (cGH) obtained from National Institute of Health (NIH), Islamabad
cDNA as a supplement agent into a mammalian expression were used for expression studies. The cells were grown in
vector, to carry out expression studies in vitro using animal Medium 199-Hepes containing 10% fetal bovine serum, 1%
cell line and then to evaluate its biological response in vivo penicillin streptomycin solution (PSS), 1% amphotericin B
using animal model. This may help to develop feasible and 0.6% L-glutamine by culturing the cells in 12 wells
plasmid based delivery system for growth hormone cloned plate (Corning, USA) and incubated in the humidified CO2
from various farming animals to enhance the production of incubator at 37oC. Cells with 80% of confluence were used
milk and meat in high demand market like South East Asian for transfection experiments using calcium phosphate co-
countries and help to raise livestock sector. precipitation method described by Wigler et al. (1978).
After transfection, the cells were incubated from 48 to 96 h.
Materials and Methods Thereafter the media and cell monolayer were harvested and
analyzed by dot blot.
Plasmid Preparations
Bioactivity Evaluation
Caprine GH-pTZR57 construct in DH5α cells with
accession # DQ 307368 by Khan et al. (2005) was available The biological activity of the construct i.e. rcGH cDNA was
in Biotechnology Laboratory, Department of Biochemistry, evaluated in white BALB/c, an albino female mice by
PMAS-Arid Agriculture University Rawalpindi. The clone weight gain and tibia epiphyseal width assays using
was refreshed from its glycerol stock and plasmid DNA was endotoxin free plasmid DNAs (Greenspan et al., 1949; Zhu
extracted by the method described by Maniatis et al. (1989). et al., 1997). Forty five, four weeks old BALB/c mice were
Similarly, mammalian expression vector pND cloned in divided into three groups containing fifteen animals each

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 Butt et al. / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

and kept at animal house facility of National Institute of

Health (NIH), Islamabad to acclimatize for one week before
inoculating with plasmid DNAs. First group of mice were
injected with 100 µg pND vector without insert in100 µL of
phosphate buffer saline (PBS), second group was injected
with 100 µL of PBS solution and third group was injected
with 100 µg rcGH-pND in 100 µL PBS of intramuscularly.
All animals were examined for change in body weight and
width of the tibial epiphysis starting from zero week till the
end of four weeks with one week interval. The data was
analyzed by using analysis of variance (ANOVA) through
MS Excel Data analysis tool Version 2007. The means with
significant difference were compared with Duncan Multiple
Range Test (DMRT) using M-STAT C.
Fig. 1: Restriction analysis of rcGH sub-cloned in pND
Results mammalian vector. Lane 1. 1 Kb ladder (Invitrogen, USA),
Lane 2-3. rcGH cDNA of ~692 bp obtained from pND
The primary objective of this study was to evaluate the constructs restricted with Eco RV
bioactivity of cloned GH cDNA from caprine breed; Beetal
using novel DNA-based expression technology.
Sub-cloning of cGH cDNA in pND Vector
The sub cloning of cGH cDNA in pND vector was
confirmed by restricting the clone with restriction enzyme;
EcoR V which generated an expected fragment of 692 bp.
The obtained result is shown in Fig. 1.
Expression Studies
Expression of cGH cDNA was done in Vero cell line. The
maximum level of expression was observed with 2.5 µg of
construct i.e., rcGH-pND at 72 h of post transfection in
cultural media. The pND without insert i.e., rcGH was used
as negative control, while commercially available GH
hormone was used as positive control in these in vitro Fig. 2: Confirmation of expression of rcGH-pND contruct
expression studies, which showed no expression and strong from cell’s culture media by dot blot. (A) The Vero cells
expression, respectively (Fig. 2). Similarly level of transfected with 2.5 µg of pND having rcGH incubated
expression, determined in cell lysate, showed no detectable from 48 h to 96 h. (B) The Vero cells transfected with 2.5
expression at any incubation hours with both concentrations µg of pND vector incubated from 48 h to 96 h. (C) The
of rcGH-pND used i.e., 2.5 µg and 5 µg (Fig. 3). Vero cells transfected with 5 µg of pND having rcGH from
48 h to 96 h. GH is commercially available growth
Activity Evaluation hormone, used as a positive control. (D) The Vero cells
transfected with 5 µg of pND vector incubated from 48 h
The bioactivity evaluation of plasmid based recombinant to 96 h
caprine growth hormone (rcGH-pND) by weight gain and
tibia width bioassays in BALB/c female mice showed that
there was significant increase in the weight (P<0.05) as well animals; ovine and caprine) using different cell lines i.e.,
as in width of tibia (P< 0.05) of the mice from zero to COS-7, Verots S3, SP2/0 respectively showed the presence
fourth week of the experiment showing gradual normal of recombinant GH protein in cultured supernatant of
growth pattern in comparison to PBS and pND control transfected cells (Min et al., 2009). The positive results of
(Table 1, 2). our expression studies in animal cell line model have
provided the bases to proceed for determining the biological
Discussion activity of rcGH-pND in animal model. Thus, the results of
weight gain and tibia width assays showed that rcGH-pND
Expression studies carried out by other investigators was biologically active in the muscle of animal and may be
using GH cDNA from other species like human and ovine used as supplement to increase the biomass and meat
(which is identical to caprine and thus can be used in both production in goat breeds as well as in sheep breeds since

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Table 1: Weight gain data of BALB/c mice from 1-4 weeks post inoculation of recombinant caprine growth hormone
(rcGH) in mammalian pND vector
Treatment 0 week 1st week 2nd week 3rd week 4th week
pND 20.5±2.12eA 23.0±1.41dA 26.5±0.71cA 30.5±0.71bB 34.5±0.71aB
PBS 19.5±0.71eA 23.5±0.71dA 26.5±2.12cA 31.0±1.41bB 33.5±3.54aB
rcGH-pND 16.5±0.71eB 19.5±0.71dB 28.0±1.41cA 34.0±1.41bA 36.5±2.12aA

Table 2: Tibia width data of BALB/c mice from 1-4 weeks post inoculation of recombinant caprine growth hormone
(rcGH) in mammalian pND vector
Treatment 0 week 1st week 2nd week 3rd week 4th week
cA bA bB aB
pND 5.28±0.97 7.36±1.40 9.69 ± 0.05 13.98±0.11 14.28±0.31aB
PBS 5.38±1.40dA 7.76±1.40cA 9.49±0.81bB 13.67±0.02aC 14.12±0.54aB
rcGH-pND 4.27± 0.15eA 6.65±0.15dA 9.03±0.21cB 15.20±0.21bA 15.69±0.16aA
Means with similar smaller superscripts in rows are insignificant (P>0.05) with each other. Means with similar capital superscripts in columns are
insignificant (P>0.05) with each other

based technology to deliver growth hormone releasing

hormone (GHRH), a releasing hormone for growth
hormone, to various animal species for screening,
toxicology and therapy. A GHRH-expressing plasmid was
engineered for efficient expression in skeletal muscle and
following intramuscular injection enhanced by
electroporation in piglets, it was observed that GHRH is
synthesized in injected muscle, secreted into blood stream
and stimulated normal pituitary GH production and release.
The release GH then in turn stimulated the growth of
piglets. In another study carried out by Meng et al. (2004)
observed enhanced growth in sheep when plasmid
expression vector having sheep GHRH was injected in
muscle tissue of the animals. These studies indirectly
support present study in which plasmid DNA of growth
Fig. 3: Confirmation of expression of rcGH-pND contruct hormone rather than plasmid DNA of GHRH has been used
from cell lysate by dot blot. (A) The Vero cells transfected to evaluate the biological activity. Thus all these studies
with 2.5 µg of pND having rcGH incubated from 48 h to (indirect) including ours (direct and first of its kind) support
96 h. (B) The Vero cells transfected with 2.5 µg of pND the notion that plasmid based gene delivery system is more
vector incubated from 48 h to 96 h. (C) The Vero cells feasible in terms of administrating plasmid rather than
transfected with 5 µg of pND having rcGH incubated from administrating the product of gene in purified form which is
48 h to 96 h. GH is commercially available growth not cost effective. However, further studies are required to
hormone, used as a positive control. (D) The Vero cells determine the effect of plasmid-based delivery of GH rather
transfected with 5 µg of pND vector incubated from 48 h than protein based delivery on milk production by farming
to 96 h animals.
In conclusion, the recombinant plasmid based
caprine growth hormone is identical to ovine (Ascacio- technology seems to be a good source of gene delivery and
Martinez and Barrera-Saldaña, 2012). However, effect of in vivo transgene expressions. Further, it is cost effective
plasmid based rcGH for enhancement of milk production therapy in comparison to exogenous administration of
needs to be determined. The weight gain and tibia width recombinant homologous GH especially in resource limited
assays has been used in a number of studies especially to setting to enhance the genetic potential of animals to
evaluate the effect of human growth hormone in dwarf little increase meat and milk production.
mice (Bellini and Bartolini, 1993), recombinant human
growth hormone (George et al., 2007; Kwak et al., 2009), Acknowledgements
recombinant DNA derived human growth
hormone/somatotropin in hypophysectomized rats (Zhu et The authors are thankful to Dr. Garry Rhodes, University of
al., 1997). The reason being that recombinant human California, Davis, CA, USA for providing mammalian
growth hormone is used to treat growth hormone deficiency expression vector pND. Further, we are highly grateful to
in children and adults and wasting in AIDS patients (Cox et the Higher Education Commission, Pakistan, for generous
al., 2007). financial assistance.
Draghia-Akli et al. (2003; 2006) also used plasmid

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 Butt et al. / Int. J. Agric. Biol., Vol. 16, No. 3, 2014

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227‒230 (Received 31 August 2013; Accepted 14 October 2013)

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