Journal of Iranian Blood Transfusion
فصلنامه پژوهشی خون
bloodj
Medical Sciences
http://bloodjournal.ir
91
journal91
1027-9520
1735-8248
10.61882/bloodj
fa
jalali
1401
9
1
gregorian
2022
12
1
19
4
online
1
fulltext
fa
کلونینگ آنزیم آلفا گالاکتوزیداز با هدف تبدیل آنتیژن B گروه خونی به آنتیژن O
Cloning and expression a-galactosidase in order to convert B to O blood group
انتقال خون
Blood Transfusion
پژوهشي
Research
<div style="text-align: justify;"><span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><span style="font-weight:bold"><span lang="FA" style="line-height:80%"><span style="font-family:Jadid"><span style="font-weight:normal">چکید</span></span></span><span lang="AR-SA" style="line-height:80%"><span style="font-family:Jadid"><span style="font-weight:normal">ه</span></span></span><span lang="FA" style="line-height:80%"><span style="font-family:Jadid"><span style="font-weight:normal"></span></span></span></span></span></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><i><span lang="FA" style="font-size:9.0pt"><span style="line-height:80%"><span style="font-family:Titr">سابقه و هدف </span></span></span></i></b></span></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">کمبود گروه‎های خونی سیستم </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">ABO</span></span></b><b><span style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> یکی از مشکلات متداول مراکز انتقال خون در دنیا می</span></span></span></b><b><span dir="LTR" lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">‎</span></span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">باشد. هم زمان با پیشرفت علم مهندسی ژنتیک و تخلیص پروتئین</span></span></span></b><b><span dir="LTR" lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">‎</span></span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">ها، تبدیل آنتی</span></span></span></b><b><span dir="LTR" lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">‎</span></span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">ژن‎های </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">A</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> و </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">B</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> به آنتی</span></span></span></b><b><span dir="LTR" lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">‎</span></span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">ژن </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">O</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> و ایجاد گروه خونی واحد مطرح شد. هدف از این پژوهش، بیان ژن آلفا گالاکتوزیداز در میزبان پروکاریوتی </span></span></span></b><b><i><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">E.coli</span></span></i></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> با ایجاد تغییرات پس از ترجمه و در نهایت تبدیل آنتی‎ژن </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">B</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> گروه خونی به آنتی‎ژن </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">O</span></span></b><b><span style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> بود.</span></span></span></b></span></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><i><span lang="FA" style="font-size:9.0pt"><span style="line-height:80%"><span style="font-family:Titr">مواد و روشها</span></span></span></i></b></span></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">در این مطالعه تجربی، ژن آلفا گالاکتوزیداز دانه قهوه که پس از بهینه‎سازی کدون‎ها در پلاسمید </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">pBHA</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> حاوی جایگاه برش آنزیم‎های </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">BamH1</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> و </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">Nco1</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> کلون شده بود، در باکتری </span></span></span></b><b><i><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">E.coli</span></span></i></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> سویه </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">TOP10</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> تکثیر و استخراج شد. ژن آلفا گالاکتوزیداز پس از برش با آنزیمهای فوق و تخلیص روی ژل آگاروز مجدداً در پلاسمید بیانی </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">pET-20b</span></span></b><b><span style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> کلون شده و به باکتری </span></span></span></b><b><i><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">E.coli</span></span></i></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> سویه </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">rosetta 2 (DE3)</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> وارد شد. بیان ژن با استفاده از القاکننده </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">IPTG</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> انجام شد. وجود پروتئین تولیدی با استفاده از </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">SDS-PAGE</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> و وسترن بلات بررسی شد. آزمون عملکرد پروتئین نیز با توانایی تبدیل گلبول‎های </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">B</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> به </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">O</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> ارزیابی شد. </span></span></span></b></span></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><i><span lang="FA" style="font-size:9.0pt"><span style="line-height:80%"><span style="font-family:Titr">یافتهها</span></span></span></i></b></span></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">وسترن بلات و </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">SDS-PAGE</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> وجود پروتئین در پری پلاسم باکتری را تائید کرد. آنزیم تخلیص شده توانست آنتی‎ژن </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">B</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> را به طور نسبی از گلبول قرمز حذف کرده و میزان آگلوتیناسیون با آنتی‎سرم </span></span></span></b><b><span dir="LTR" style="font-size:9.0pt"><span style="line-height:80%">B</span></span></b><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:"> را به میزان زیادی کاهش دهد. </span></span></span></b></span></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><i><span lang="FA" style="font-size:9.0pt"><span style="line-height:80%"><span style="font-family:Titr">نتیجه گیری</span></span></span></i></b></span></span></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:80%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span lang="FA" style="font-size:11.0pt"><span style="line-height:80%"><span 2="" lotus="" style="font-family:">بیان پروتئین یوکاریوتی در میزبان پروکاریوتی می‎تواند در تولید انبوه آنزیم گالاکتوزیداز برای کاربرد در مراکز انتقال خون کارگشا باشد. بهینهسازی شرایط مختلف بیان برای دستیابی به مقادیر کافی از آنزیم ضروری می‎باشد.</span></span></span></b></span></span></span></span></span></span><br>
</div>
<div dir="rtl" style="text-align: left;"><span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><span style="font-size:12.0pt">Abstract</span></b><b><span style="font-size:12.0pt"></span></b></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><i>Background and Objectives</i></b></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times="">The shortage of different ABO blood groups is a permanent problem that many blood transfusion centers encounter. The conversion of blood group A and B to group O is a solution to this problem. In this research we aimed to clone and express the α-galactosidase enzyme gene to convert the blood group B to O.</span></span></span></span></span><br>
<br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><i>Materials and Methods</i></b></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times="">In this experimental study, the α-galacosidase gene from coffee bean was codon-optimized and cloned into the pBHA plasmid. After replication in <i>E. coli</i>, plasmid was cut with BamH1 and Nco1 restriction enzymes and the α-galacosidase gene was purified by gel electrophoresis. The gene was then cloned into the expression vector, pET-20b. The expression construct was introduced into E. coli Rosetta 2 (DE3) and the expression of the enzyme was induced by IPTG. The presence of protein production was investigated using SDS-PAGE and Western blotting. Protein function test was also evaluated by the ability of B cells to convert to O cells.</span></span></span></span></span><br>
<br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><i>Results</i></b></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times="">The presence of the recombinant protein was confirmed by SDS-PAGE and Western blot assay. The purified protein could partially convert blood group B RBCs into group O as detected by decreasing the agglutination strength with B antiserum from 4+ to less than 1+.</span></span></span></span></span><br>
<br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times=""><b><i>Conclusions </i></b></span></span></span></span></span><br>
<span style="font-size:10pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="unicode-bidi:embed"><span new="" roman="" style="font-family:" times="">The production of eukaryotic proteins in a prokaryotic host is a major step in mass production. Optimization of different expression conditions is essential to achieve sufficient amount of enzyme.</span></span></span></span></span><br>
<br>
</div>
کلمات کلیدی: گروه خونی, گالاکتوزیداز, E.coli
Key words: Blood Group, Galactosidase, E coli
301
312
http://bloodjournal.ir/browse.php?a_code=A-10-1509-1&slc_lang=fa&sid=1
A.
Hadad deilami
افسانه
حداد دیلمی
9100319475328460023363
9100319475328460023363
No
دانشکده علوم زیستی دانشگاه آزاد اسلامی واحد تهران شمال
M.
Salehi
میترا
صالحی
mitra_salehi_microbiology@yahoo.com
9100319475328460023364
9100319475328460023364
Yes
استادیار دانشکده علم زیستی دانشگاه آزاد اسلامی واحد تهران شمال
M.
Shahabi
مجید
شهابی
9100319475328460023365
9100319475328460023365
No
استادیار مرکز تحقیقات انتقال خون ـ مؤسسه عالی آموزشی و پژوهشی طب انتقال خون