Prokaryotic Expression of the Influenza A (H5N1) Neuraminidase

(1)

⁹⁴

Prokaryotic Expression of the Influenza A (H5N1) Neuraminidase

Zahra Latif^1,2, Behrokh Farahmand³,

Maryam Saleh⁴, Zahra Barghi⁵

1MSc in Molecular Biotechnology, Malek Ashtar University of Technology, Tehran, Iran

2Department of Influenza Virus Research and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Iran

3Assistant Professor, Department of Influenza Virus Research and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Iran

4PhD in Medical Microbiology, Department of Influenza Virus Research and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Iran

5MSc in Biochemistry, Department of Influenza Virus Research and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Iran

(Received November 15, 2020 ; Accepted February 1, 2021)

Abstract

Background and purpose: Highly Pathogenic Influenza A(H5N1) viruses cause vast economic losses throughout the world. They may circulate in animals and be able to spread from human to human.

Therefore, launching diagnostic tests are highly essential to control the influenza infection. Studies on the amino acid sequence of the neuraminidase (NA) protein of influenza viruses revealed that NA is the most immunogenic protein in naïve animals which can simply stimulate the humoral immune system well.

Materials and methods: Influenza virus NA gene (A/Indonesia/5/2005(H5N1) was cloned into pET21a and expressed in host E. coli (BL21) strains. Then, the expression level of NA protein was optimized for different IPTG inductor concentrations and times.

Results: Findings showed the integrity of pET21a-NA construct. Emerging bands with the expected molecular weight (38KDal) on SDS-PAGE and WB analysis confirmed the successful expression of target protein in E.coli BL21 strain. In silico analysis showed integrity of major epitopes in the structure of fused version of NA produced in this work.

Conclusion: The new recombinant NA has the potential to be used directly in serological tests.

It could be also used in polyclonal antibody preparation which is employed as an essential material in western blot analyses and other immunological and serological studies, such as ELISA, immunocytochemistry, and immunohistochemistry.

Keywords: neuraminidase, prokaryote system, diagnostic tests, Influenza A (H5N1) virus

J Mazandaran Univ Med Sci 2021; 31 (195): 94-102 (Persian).

* Corresponding Author: Behrokh Farahmand - Pasteur Institute of Iran, Tehran, Iran (E-mail: zfotoukian@gmail.com)

[ Downloaded from jmums.mazums.ac.ir on 2022-10-31 ]

(2)

هرود ﮑﯾ و ﯽﺳ هرﺎﻤﺷ ﻢ 195 ﻦﯾدروﺮﻓ لﺎﺳ 1400 ) 102 - 94 (

ياﺰﻧاﻮﻠﻔﻧآ سوﺮﯾو زاﺪﯿﻨﯿﻣآرﻮﻧ نژ ﯽﺘﻧآ ﯽﺗﻮﯾرﺎﮐوﺮﭘ نﺎﯿﺑ A

(H5N1)

ﯿﻄﻟ اﺮﻫز ﻒ

1 و

2

ﺪﻨﻤﻫﺮﻓ خﺮﻬﺑ 3

ﺮﻣ ﯾ ﻢ ﺢﻟﺎﺻ 4

ﻗﺮﺑ اﺮﻫز ﯽ 5

هﺪﯿﮑﭼ

ﻪﻘﺑﺎﺳ و :فﺪﻫ سوﺮﯾو اﺰﻧاﻮﻠﻔﻧآ ي دﺎﺣ قﻮﻓ نﺎﮔﺪﻧﺮﭘ A (H5N1) رد يدﺎــﯾز يدﺎــﺼﺘﻗا تارﺎﺴﺧ دﺎﺠﯾا ﺚﻋﺎﺑ ﺮــﺳاﺮﺳ

رد ﺖﺳا ﻦﮑﻤﻣ و ﺪﻧﻮﺷ ﯽﻣ نﺎﻬﺟ ﺣ

.ﺪــﻨﮐ اﺪــﯿﭘ نﺎــﺴﻧا ﻪﺑ نﺎﺴﻧا زا رﺎﺸﺘﻧا ﯽﯾﺎﻧاﻮﺗو ﺪﺷﺎﺑ شدﺮﮔرد تﺎﻧاﻮﯿ هار ﻦﯾاﺮﺑﺎــﻨﺑ

يزاﺪــﻧا

ﺖﺴﺗ .ﺖــﺳا يروﺮــﺿ اﺰﻧاﻮــﻠﻔﻧآ ﺖــﻧﻮﻔﻋ لﺮــﺘﻨﮐ ياﺮــﺑ ﯽــﺼﯿﺨﺸﺗ يﺎﻫ ﻦﯿﺌﺗوﺮــﭘ ﻪــﻨﯿﻣآ يﺎﻫﺪﯿــﺳا ﯽﻟاﻮــﺗ درﻮــﻣ رد تﺎــﻌﻟﺎﻄﻣ

) زاﺪﯿﻨﯿﻣآرﻮﻧ اﺰﻧاﻮﻠﻔﻧآ سوﺮﯾو (NA

ﻪﮐ ﺖﺳا هداد نﺎﺸﻧ ﯽﻨﻤﯾاNA

از ﻦﯿﺌﺗوﺮﭘ ﻦﯾﺮﺗ د

ﯽــﻣ و ﺖــﺳا تﺎــﻧاﻮﯿﺣ ر ﻢﺘــﺴﯿﺳ ﺪــﻧاﻮﺗ

.ﺪﯾﺎﻤﻧ ﮏﯾﺮﺤﺗ ار لارﻮﻣﻮﻫ ﯽﻨﻤﯾا

شور و داﻮﻣ :ﺎﻫ

ﯽــﺑﺮﺠﺗ ﻪﻌﻟﺎﻄﻣ ﻦﯾا رد اﺰﻧاﻮــﻠﻔﻧآ سوﺮــﯾو

) A/Indonesia/5/2005: H5N1 رد (

pET21a رد و نﻮــﻠﮐ

) نﺎﺑﺰﯿﻣ (BL21 E. coli نﺎﯿﺑ ناﺰﯿﻣ ﺲﭙﺳ .ﺪﺷ نﺎﯿﺑ ﺮﮔﺎﻘﻟا ﺖﻈﻠﻏ يﺎﻫﺮﯿﻐﺘﻣ ﺮﻈﻧ زاNA

وIPTG ﻪﻨﯿﻬﺑ نﺎﻣز ﺎﺳ .ﺪﺷ يز

ﻪﺘﻓﺎﯾ :ﺎﻫ

،ﺞﯾﺎﺘﻧ رﺎﺘﺧﺎﺳ ﺖﺤﺻ Pet21a-NA

رﺎــﻈﺘﻧادرﻮﻣ ﯽﻟﻮــﮑﻟﻮﻣ نزو ﺎــﺑ هﺪــﺷﺮﻫﺎﻇ يﺎﻫﺪــﻧﺎﺑ .داد نﺎﺸﻧ ار KDal)

38 (

يور SDS-PAGE ﺖﯿﻘﻓﻮﻣ ﯽﻧﺎﯿﺑ ﻪﮐ ﺪﺷ ﺪﯿﯾﺎﺗ تﻼﺑ نﺮﺘﺳو ﺎﺑ

زا ﺰﯿﻣآ ﻪﯾﻮﺳرد فﺪﻫ ﻦﯿﺌﺗوﺮﭘ E. coli BL21

و ﻪﯾﺰﺠﺗرد .دﻮﺑ

ﻪﻧﺎﯾار نورد ﻞﯿﻠﺤﺗ ﯽﭘا ﯽﺘﺳرد ،يا

هﺪﻤﻋ پﻮﺗ ﻪﺨﺴﻧ رﺎﺘﺧﺎﺳ رد

هﺪﺷ ﺪﯿﻟﻮﺗNA ﻦﯾارد

ﻪﻌﻟﺎﻄﻣ داد نﺎﺸﻧ ار .

:جﺎﺘﻨﺘﺳا ﯽﻣ ﺪﯾﺪﺟ ﺐﯿﮐﺮﺗﻮﻧNA

ﻪﺑ ﺪﻧاﻮﺗ يژﻮﻟوﺮﺳ تﺎﺸﯾﺎﻣزآ رد ﻢﯿﻘﺘﺴﻣ رﻮﻃ ﻦﯿــﻨﭽﻤﻫ .دﻮــﺷ هدﺎﻔﺘــﺳا ﺺﯿﺨــﺸﺗ ياﺮﺑ

ﯽﻣ ﯽﺘﻧآ ﻪﯿﻬﺗ ضﺮﻌﻣ رد ﺪﻧاﻮﺗ رد ﯽﺳﺎﺳا هدﺎﻣ ناﻮﻨﻋ ﻪﺑ ﻪﮐ ،دﺮﯿﮔراﺮﻗ لﺎﻧﻮﻠﮐ ﯽﻠﭘ يدﺎﺑ

و ﻪﯾﺰﺠﺗ تﻼــﺑ نﺮﺘــﺳو ﻞﯿﻠﺤﺗ و

ﺮﯾﺎــﺳ

ﺪﻨﻧﺎﻣ ﮏﯾژﻮﻟﻮﻧﻮﻤﯾا تﺎﻌﻟﺎﻄﻣ ELISA

.ﺖﺳا هﺪﺷ هدﺎﻔﺘﺳا ﯽﻤﯿﺷﻮﺘﯿﺳﻮﻧﻮﻤﯾا ،

:يﺪﯿﻠﮐ يﺎﻫ هژاو ﺖﺴﺗ ،تﻮﯾرﺎﮐوﺮﭘ ﻢﺘﺴﯿﺳ ،زاﺪﯿﻨﯿﻣآرﻮﻧ

اﺰﻧاﻮﻠﻔﻧآ سوﺮﯾو ،ﯽﺼﯿﺨﺸﺗ يﺎﻫ ي

)A (H5N1

ﻪﻣﺪﻘﻣ

ياراد اﺰﻧاﻮﻠﻔﻧآ يرﺎﻤﯿﺑ ﺖﯿﻤﻫا

ﯽﻧﺎﻬﺟ هدﻮﺑ و ﻪﻧ ﺎﻬﻨﺗ

ﺖﯿﻌﻤﺟ رد يﺎﻫ

ﯽﻧﺎﺴﻧا ﻪﮑﻠﺑ رد و نﺎﮔﺪﻧﺮﭘ تﺎﻧاﻮﯿﺣ

ردﺎــﻗ

ﻪﺑ شﺮﺘﺴﮔ ﯽﻣ ﺪــﺷﺎﺑ ) 2،1 ﻢــﻬﻣ ﻪــﻠﻤﺟزا .( ﻦﯾﺮــﺗ

ﻦﯿﺌﺗوﺮــﭘ يﺎــﻫ

ﯽﺤﻄﺳ ﯽــﻣ سوﺮــﯾو ناﻮــﺗ

ﻪــﺑ زاﺪــﯿﻨﯿﻣآرﻮﻧ هرﺎــﺷا(NA)

دﻮﻤﻧ ﻪﮐ ﻪﺑ ترﻮﺻ يور يﺪﯾاوز ﺶﺷﻮﭘ

يﺪــﯿﭙﯿﻟ نﺎــﯾﺎﻤﻧ ﯽﻣ

دﻮﺷ )1 ﻦﯿﻟوا .( ﺦﺳﺎﭘ ﻪﺑ ﺖﺒﺴﻧ ﯽﻨﻤﯾا اﺰﻧاﻮــﻠﻔﻧآ سوﺮــﯾو

ي

عﻮﻧ ردA نﺪﺑ ﯽــﭘرد اﺰﻧاﻮــﻠﻔﻧآ ﺖــﻧﻮﻔﻋ زا

ﻖــﯾﺮﻃ ﺪــﯿﻟﻮﺗ

ﯽﺘﻧآ يدﺎﺑ ﺎــﻫ ﻪــﯿﻠﻋﺮﺑ زاﺪــﯿﻨﯿﻣآرﻮﻧ ترﻮــﺻ

ﯽــﻣ دﺮــﯿﮔ )3 . (

لﻮﻠــﺳ زا سوﺮــﯾو نﺪﺷ ﺎﻫر ﺐﺟﻮﻣ زاﺪﯿﻨﯿﻣآرﻮﻧ هدﻮــﻟآ يﺎــﻫ

و ﺖﻧﻮﻔﻋ ﻪﺑ لﻮﻠﺳ نﺪﺷﺮﯿﮔرد

ﯽﻣ ﻢﻟﺎﺳ يﺎﻫ دﻮﺷ

ﻦﯾاﺮﺑﺎﻨﺑ

:لﻮﺌﺴﻣ ﻒﻟﻮﻣ ﺪﻨﻤﻫﺮﻓ خﺮﻬﺑ

ناﺮﻬﺗ- : رﻮﺘﺳﺎﭘ نﺎﺑﺎﯿﺧ

، كﻼﭘ 69 ، ﻮﺘﯿﺘﺴﯿﻧا رﻮﺘﺳﺎﭘ ناﺮﻬﺗ

farahmand.b2@gmail.com E-mail:

1 . ﺪﺷرا ﯽﺳﺎﻨﺷرﺎﮐ ﯽﻟﻮﮑﻟﻮﻣ يژﻮﻟﻮﻨﮑﺗﻮﯿﺑ

، مﻮﻠﻋ هﺪﮑﺸﻫوﮋﭘ

،ﺮﺘﺷا ﮏﻟﺎﻣ ﯽﺘﻌﻨﺻ هﺎﮕﺸﻧاد ،ﯽﺘﺴﯾز يروﺎﻨﻓو ناﺮﯾا ،ناﺮﻬﺗ

2 . ﻮﺘﯿﺘﺴﻧا ،ﯽﺴﻔﻨﺗ يﺎﻫ سوﺮﯾو ﺮﯾﺎﺳو اﺰﻧﻻﻮﻔﻧآ ﺶﺨﺑ رﻮﺘﺳﺎﭘ

،ناﺮﻬﺗ

،ناﺮﻬﺗ ناﺮﯾا

3 رﺎﯾدﺎﺘﺳا . ، ﻮﺘﯿﺘﺴﻧا ،ﯽﺴﻔﻨﺗ يﺎﻫ سوﺮﯾو ﺮﯾﺎﺳو اﺰﻧﻻﻮﻔﻧآ ﺶﺨﺑ ﻮﺘﺳﺎﭘ

ر

،ناﺮﻬﺗ

4 . يﺮﺘﮐد ،يژﻮﻟﻮﯿﺑوﺮﮑﯿﻣ ﻮﺘﯿﺘﺴﻧا ،ﯽﺴﻔﻨﺗ يﺎﻫ سوﺮﯾو ﺮﯾﺎﺳو اﺰﻧﻻﻮﻔﻧآ ﺶﺨﺑ

رﻮﺘﺳﺎﭘ

،ناﺮﻬﺗ

5.

ﺪﺷرا ﯽﺳﺎﻨﺷرﺎﮐ ،ﯽﻤﯿﺷﻮﯿﺑ

ﻮﺘﯿﺘﺴﻧا ،ﯽﺴﻔﻨﺗ يﺎﻫ سوﺮﯾو ﺮﯾﺎﺳو اﺰﻧﻻﻮﻔﻧآ ﺶﺨﺑ رﻮﺘﺳﺎﭘ

،ناﺮﻬﺗ

: ﺖﻓﺎﯾرد ﺦﯾرﺎﺗ 25

/8 / 1399 رﺎﺗ : تﺎﺣﻼﺻا ﺖﻬﺟ عﺎﺟرا ﺦﯾ 3/

9 / 1399 : ﺐﯾﻮﺼﺗ ﺦﯾرﺎﺗ 13

/ 11 / 1399

(3)

ﺑﯿ نﺎ رﺎﮐوﺮﭘ ﯾ ﺗﻮ ﯽ وﯾ سوﺮ اﺰﻧاﻮﻠﻔﻧآ ي A

96 ﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ﻪﻠﺠﻣ نارﺪﻧزﺎﻣ ﯽﮑ

هرﺎﻤﺷ ،ﻢﮑﯾ و ﯽﺳ هرود

195 ﻦﯾدروﺮﻓ ، 1400

ﺪﯾﺪﻧﺎﮐ ﺖﺴﺗ ياﺮﺑ ﯽﺒﺳﺎﻨﻣ ﯽﺼﯿﺨﺸﺗ يﺎﻫ

ﻦﺴﮐاو و .ﺖﺳا ﺎﻫ

ﯽــﺑﺮﺠﺗ ﺪﻫاﻮــﺷ رد

درﻮــﻣ سوﺮــﯾو يﺎــﻫ اﺰﻧاﻮــﻠﻔﻧآ نﺎــﺸﻧ

ﯽﻣ ﺪﻫد ﻦــﮑﻤﻣ زاﺪــﯿﻨﯿﻣآرﻮﻧ نﻮﯿــﺳﻼﯾزﻮﮑﯿﻠﮔ ﻪﮐ ﺖــﺳا

ياﺮﺑ ﻞﮑﺷ يﺮﯿﮔ ﺐﺳﺎﻨﻣ ﯽﯾﺎﺳﺎﻨﺷ و هﺪﻧﺮﯿﮔ

نﺎﺑﺰﯿﻣ ﻢــﻬﻣ

،ﺪﺷﺎﺑ ﺎﻣا رد ﯽﻨﻤﯾا ﯽﯾاز نآ ﺖﯿﻤﻫا

،دراﺪــﻧ ﯽﻧاﺪــﻨﭼ ﻦﯾاﺮﺑﺎــﻨﺑ

ﯽﻧﺎﻣز ﻪﮐ رد ﻢﺘﺴﯿﺳ ﯽﺗﻮﯾرﺎﮐوﺮﭘ نﺎﯿﺑ

،دﻮﺷ صاﻮــﺧ نﺎــﻤﻫ

ﯽﻧژ ﯽﺘﻧآ دﻮﺧ ار ﻆﻔﺣ ﯽﻣ نﺎﯿﺑ .ﺪﻨﮐ تﺎﻌﻄﻗ ردزاﺪــﯿﻨﯿﻣآرﻮﻧ

ﻢﺘﺴﯿﺳ ﯽﺗﻮﯾرﺎﮐوﺮﭘ ﻪﺑ

رﻮﻃ هﻮﻘﻟﺎﺑ ﯽﻣ ﺪﻧاﻮﺗ دﺮــﺒﻫار رﺎﯿــﺴﺑ

يﺮﺛﻮــﻣ ﺪــﯿﻟﻮﺗ ياﺮــﺑ ﺮﯾدﺎــﻘﻣ

يدﺎــﯾز زا زاﺪــﯿﻨﯿﻣآرﻮﻧ رد

تﺪﻣ ﯽﻧﺎﻣز هﺎﺗﻮﮐ ﺖﯿﮐ رد هدﺎﻔﺘﺳا ﺖﻬﺟ يﺎــﻫ

و ﯽــﺼﯿﺨﺸﺗ

ﻦﺴﮐاو صﻮﺼﺧ ﻪﺑ ﺎﻫ ناﺮﯾا رد

سوﺮــﯾو ﻦــﯾا غاد ﻪــﻄﻘﻧ ﻪﮐ)

ﯽﻣ بﻮﺴﺤﻣ ﺪﺷﺎﺑ (دﻮﺷ

) 5،4 ﻦﯿﻨﭽﻤﻫ .( ﻖﯾرﺰﺗ زاﺪﯿﻨﯿﻣآرﻮﻧ

ﺺﻟﺎﺧ هﺪﺷ ﻪﺑ شﻮﻣ ﺐﺟﻮﻣ يﺎﻘﻟا ﺪﯿﻟﻮﺗ ﯽﺘﻧآ يدﺎﺑ ﺪﺿ

ﺖﯿﻟﺎﻌﻓ نﻮﯿﺳﺎﻨﯿﺗﻮﻠﮔﺎﻤﻫ هﺪﺷ

و رد ﺖﯾﺎﻬﻧ ﺮﺠﻨﻣ ﻪﺑ زا ﻦﯿﺑ

ﻦﺘــﻓر اﻮﺗ ﯽﯾﺎــﻧ سوﺮــﯾو رد دﺎــﺠﯾا ﺖــﻧﻮﻔﻋ ﯽــﻣ دﻮــﺷ )5 .(

نﺎﮔﺪﻧﺮﭘدﺎﺣ قﻮﻓ سوﺮﯾو يرﺎﻤﯿﺑ ﺮﺑ هوﻼﻋH5N1

ﯽــﯾاز

يرﺎﻤﯿﺑ ﺚﻋﺎﺑ نﺎﮔﺪﻧﺮﭘ رد ﺰــﯿﻧ تﺎــﻧاﻮﯿﺣو نﺎــﺴﻧا رد ﯽﯾاز

ﯽﻣ ﺖﺳا هﺪﺷ هﺪﻫﺎﺸﻣ ﻢﻫ نﺎﺴﻧا ﻪﺑ نﺎﺴﻧا لﺎﻘﺘﻧا و دﻮﺷ )6

،(

عاﻮــﻧا زا سوﺮــﯾو عﻮﻧ ﻦﯾا يور ﺮﺑ ﺮﺿﺎﺣ ﻪﻌﻟﺎﻄﻣ ﻦﯾاﺮﺑﺎﻨﺑ ﺮﮔ ترﻮــﺻ اﺰﻧاﻮــﻠﻔﻧآ فﺪــﻫ ﺎــﺑ و ﺖــﻓ

ﻦﯿﺌﺗوﺮــﭘ ﺪــﯿﻟﻮﺗ

ﻢﺘﺴﯿﺳ رد زاﺪﯿﻨﯿﻣآرﻮﻧ ﺐﯿﮐﺮﺗﻮﻧ ﯽﺗﻮﯾرﺎﮐوﺮﭘ

نﺎﻣز تﺪﻣرد

ﺖﺴﺗرد هدﺎﻔﺘﺳا ﺖﻬﺟ هﺎﺗﻮﮐ ﯽﺼﯿﺨﺸﺗ يﺎﻫ

.ﺪﯾدﺮﮔ مﺎﺠﻧا

ﺎﻫ شور و داﻮﻣ

ﺰﯿﻟﺎـــــﻧآ اﺪـــــﺘﺑا ﯽﮑﯿﺗﺎـــــﻣرﻮﻔﻧاﻮﯿﺑ

يور ﯽﻟاﻮـــــﺗ

و ﯽﻣﻮﻧژ ﻪﻨﯿﻣآﺪﯿــﺳا يا

ﻦﯿﺌﺗوﺮــﭘ و ﺖــﻓﺮﮔ ترﻮــﺻ NA

ﯽﻟاﻮــــﺗ يﺪــــﯿﺗﻮﺌﻠﮐﻮﻧ نژ

سوﺮــــﯾوNA ﻔﻧآ

اﺰﻧاﻮــــﻠ

) (A/Indonesia/5/2005:H5N1 ﯽــــــﺳﺮﺘﺳد ﺪــــــﮐﺎﺑ

EU146623.1 هداد هﺎﮕﯾﺎﭘ زا

ﺑﻪNCBI ﺖﺳد ﺪﻣآ )7 ﯽﻟاﻮﺗ .(

ﺖﻬﺟزا ﺮﻈﻧدرﻮﻣ يﺪﯿﺗﻮﺌﻠﮐﻮﻧ ﻖﻃﺎﻨﻣ

ﺮﺛا ﻢﯾﺰﻧآ يﺎﻫ شﺮﺑ

هﺪﻨﻫد رد مﺮﻧ راﺰﻓا NEBcutter(v2-0) ﻪــﺑ

رﻮــﻈﻨﻣ ــﯾ ﻦﺘﻓﺎ

ﻢﯾﺰﻧآ ﺐﺳﺎﻨﻣ يﺎﻫ ﺖﻬﺟ

نﻮﻠﮐ ندﺮﮐ نژ ﺮــﻈﻧ درﻮــﻣ رد

ﺪﯿﻤﺳﻼﭘ pET21a درﻮﻣ ﯽــﺳرﺮﺑ راﺮــﻗ ﺖــﻓﺮﮔ ) 9،8 . ( ﻦــﯾا ﻪﻌﻟﺎﻄﻣ

رد ﯽﺑﺮﺠﺗ ﺶﺨﺑ

اﺰﻧاﻮﻠﻔﻧآ ي ناﺮــﯾا رﻮﺘﺳﺎﭘ ﻮﺘﯿﺘﺴﻧا

ﺪﯿﻤــﺳﻼﭘ يور ﺮــﺑ pFastBacIHNM1

هﺎﮕــﺸﻧاد زا ﻪــﮐ

دﻮﺑ هﺪﺷ ﻪﺘﻓﺮﮔ ﺮﺘﺷا ﮏﻟﺎﻣ )

10 .ﺪﺷ مﺎﺠﻧا(

ﺪﻨﯾاﺮﻓ رﻮﻈﻨﻣ ﻪﺑPCR

ﺮﯿﺜﮑﺗ نژ رد ﻪــﮐ زاﺪﯿﻨﯿﻣآرﻮﻧ

ﺪﯿﻤﺳﻼﭘ pFastBacIHNM1 ﺎــﻫﺮﻤﯾاﺮﭘ ﻂﺳﻮﺗ ﺖﺷاد راﺮﻗ

ي

ﯽﺻﺎﺼﺘﺧا مﺎﺠﻧاNA

ﺪﺷ هرﺎﻤــﺷ لوﺪﺟ) 1

ياﺮــﺑ .(

ﯽــﺣاﺮﻃ

ﺮــﻤﯾاﺮﭘ زا مﺮــﻧ راﺰــﻓا Gene Runner 6.5.51)

version (

ﯽــﮔﮋﯾو و ﺪــﺷ هدﺎﻔﺘــﺳا ﻦــﯾا ﯽــﮑﯾﺰﯿﻓ و ﯽﻤﯿــﺷﻮﯿﺑ يﺎــﻫ

ﺎﻨﯿﺳ ﺖﮐﺮﺷ ﻪﺑ ﺰﺘﻨﺳ ياﺮﺑ ﺲﭙﺳ ،ﺪﯾدﺮﮔ ﯽﺳرﺮﺑ ﺎﻫﺮﻤﯾاﺮﭘ .ﺪﺷ هدﺎﺘﺳﺮﻓ نژ

هرﺎﻤﺷ لوﺪﺟ

ﺖﮐﺮﺷ ﻂﺳﻮﺗ هﺪﺷ ﺖﻓﺎﯾرد يﺎﻫﺮﻤﯾاﺮﭘ ﯽﻟاﻮﺗ 1:

نژﺎﻨﯿﺳ

ﺮﻤﯾاﺮﭘ مﺎﻧ ﺮﻤﯾاﺮﭘ ﯽﻟاﻮﺗ

%GC

بوذ يﺎﻣد ) هزاﺪﻧا (bp

F-BamH1 5′- CTAGGATCCATGATCTCAATATG 1/

39 08 / 57 23

R-Xho1 5′- TATCTCGAGCATTTCAAAGCC 9/

42 92 / 55 21

ﺎﻫ ﻪﺘﻓﺎﯾ

ﯽﻧﺎﻣز ﻪﻣﺎﻧﺮﺑ و

ﯽﯾﺎﻣد ﺶﻨﮐاو ﺶﻨــﮐاو طﻮﻠﺨﻣ وPCR

هرﺎﻤــﺷ لواﺪــﺟ رد ﺐــﯿﺗﺮﺗ ﻪــﺑPCR 2

و 3 .ﺖــﺳا هﺪــﻣآ

master mix ياﺮﺑ

ﻞﻣﺎﺷ ﺰﯿﻧPCR 20 ﺮﺘﯿﻟوﺮﮑﯿﻣ dNTPs

،

100 ﺮﻓﺎــﺑ ﺮــﺘﯿﻟوﺮﮑﯿﻣ )PCR

،(10x 30 ﺮــﺘﯿﻟوﺮﮑﯿﻣ Mgcl2

) 25

،(رﻻﻮﻣ ﯽﻠﯿﻣ 350

.دﻮــﺑ ﻞﯾﺮﺘــﺳا ﺮﻄﻘﻣ بآ ﺮﺘﯿﻟوﺮﮑﯿﻣ

ﺶﻨﮐاو ﺖﻗر ردPCR

100 /1 زا .ﺪﺷ مﺎﺠﻧا ﻮﮕﻟاDNA

هرﺎﻤﺷ لوﺪﺟ 2

ﺎﻣد ﻪﻣﺎﻧﺮﺑ :

ﯽﯾ درﻮﻣ ﯽﻧﺎﻣزو هدﺎﻔﺘﺳا

ياﺮﺑ ﺮﯿﺜﮑﺗ نژ

NA

رد ﺶﻨﮐاو PCR

ﻪﻠﺣﺮﻣ ﺎﻣد

(داﺮﮔ ﯽﺘﻧﺎﺳ ﻪﺟرد) نﺎﻣز ﻪﺧﺮﭼ داﺪﻌﺗ

ﯽﮕﺘﺷﺮﺳاو ﻪﯿﻟوا 94

5

ﻪﻘﯿﻗد 1

ﯽﮕﺘﺷﺮﺳاو 94

30

ﻪﯿﻧﺎﺛ 35

لﺎﺼﺗا 47

30

ﻪﯿﻧﺎﺛ

شﺮﺘﺴﮔ 72

2

ﻪﻘﯿﻗد

ﯽﯾﺎﻬﻧ شﺮﺘﺴﮔ 72

5

ﻪﻘﯿﻗد 1

ﺶﻨﮐاو طﻮﻠﺨﻣ:

PCR

هدﺎﻔﺘﺳا درﻮﻣ داﻮﻣ (ﺮﺘﯿﻟوﺮﮑﯿﻣ) ﺮﻈﻧدرﻮﻣ ﺮﯾدﺎﻘﻣ

ﺤﻣ يرﺎﮐ لﻮﻠ ) master mix ( 5/

12

ﻮﮕﻟاDNA 1

وﺮﺸﯿﭘ ﺮﻤﯾاﺮﭘ 1

سﻮﮑﻌﻣ ﺮﻤﯾاﺮﭘ 1

ﻞﯾﺮﺘﺳا ﺮﻄﻘﻣ بآ 4

ﻢﯾﺰﻧآ Taq 5/

0

(4)

ﺺﻟﺎﺧ لﻮﺼﺤﻣ يزﺎﺳ جاﺮﺨﺘﺳا ﺖﯿﮐ زا هدﺎﻔﺘﺳا ﺎﺑPCR

ﺖﮐﺮﺷDNA QIAquick®

PCR Purification Kit

مﺎــﺠﻧا

ﺪﺷ ياﺮﺑ. ﻪﻧﺎﺴﻤﻫ مﺎﺠﻧا يزﺎﺳ

رد رﻮﺘﮐو PGEM-TEasy

،

ﺶﻨﮐاو ﮕﯿﻟ ﻦﯿﺑ نﻮﯿﺳﺎ لﻮﺼﺤﻣ

و هﺪــﺷ هدﺎــﻣآPCR رﻮــﺘﮐو

يﺮــﯿﺜﮑﺗ PGEM-Teasy )

PGEM-TEasy Promega

REF.A137A Lot.No.0000287313

ﻦﯾا ﻪﺑ ﻪﺟﻮﺗ ﺎﺑ ( ﻪــﮐ

هزاﺪﻧا رﻮﺘﮐو 3000 زﺎﺑﻮﻠﯿﮐ ﯽﻣ ﺪﺷﺎﺑ و ﻻﻮــﻤﻌﻣ ياﺮــﺑ ﺮــﻫ

ﺶﻨــﮐاو ligation ﻞﻗاﺪــﺣ 50 مﺮﮔﻮﻧﺎــﻧ زا رﻮــﺘﮐوT مزﻻ

ﯽﻣ ﺪﺷﺎﺑ و رد ﺶﯿﺑ ﺮﺗ تﺎﻌﻟﺎﻄﻣ ﺖﺒﺴﻧ ﯽﻟﻮﻣ ﻪﻌﻄﻗ نژ فﺪــﻫ

ﻪﺑ رﻮﺘﮐو ﺖﺒﺴﻧ 3 ﻪﺑ 1 هدﻮﺑ هرﺎﻤــﺷ لوﺪــﺟ ﻖﺒﻃ ،ﺖﺳا 4

ﺖﻬﺟ .ﺖﻓﺮﮔ مﺎﺠﻧا لﺎﺼﺗا

نآ ﺎﻫ ﻪــﺑ ﯾ ﺮﮕﯾﺪــﮑ زا ﻢﯾﺰــﻧآ

T4DNA Ligase ﺖﮐﺮﺷ

Fermentase .ﺪﺷ هدﺎﻔﺘﺳا

داﻮﻣ:

و ﺮﯾدﺎﻘﻣ زﺎﯿﻧدرﻮﻣ زا ﺎﻫ نآ ياﺮﺑ ﻪﯿﻬﺗ طﻮﻠﺨﻣ

ﺶﻨﮐاو لﺎﺼﺗا نژ ﻪﺑ رﻮﺘﮐوT

داﻮﻣ درﻮﻣ زﺎﯿﻧ راﺪﻘﻣ

ﺮﺘﯿﻟوﺮﮑﯿﻣ) (

D.W 4

PGEM-T-easy Vector(50ng) 5/

0

T4 DNA ligase 1

Sample (PCR product) 5/

0

2x rapid ligation buffer 4

ﺲﭙﺳ Tvector-NA ﺑﻪ

نﺎﺑﺰﯿﻣ ﯽﯾﺎــﯾﺮﺘﮐﺎﺑ TOP10f'

ﺪﻌﺘﺴﻣ ودﺮــﮐ اﺪــﯿﭘ لﺎﻘﺘﻧا هﺪﺷ ﺖــﯿﻠﭘ رد

يﺎــﻫ LB-Agar

ياراد ﯽــﭙﻣآ

،ﻦﯿﻠﯿــﺳ X-Gal و IPTG ﺖــﺸﮐ داد ه ،ﺪــﺷ

ياﺮﺑ ﯽﯾﺎﺳﺎﻨﺷ ﺪﯿﻤﺳﻼﭘ يﺎﻫ ﺐﯿﮐﺮﺗﻮﻧ زا ﯽﻨﻠﮐ يﺎﻫ ﺪــﺷر

هدﺮﮐ ﺮﺑ يور ﺖﯿﻠﭘ ﺖﺴﺗ ﺎﻫ rapid minipreb .ﺪــﺷ مﺎﺠﻧا

ﺲﭙﺳ زا ﯽﻨﻠﮐ ﯽﯾﺎﻫ ﺞﯾﺎﺘﻧ ﻪﮐ نآ ﺎﻫ ﺖﺒﺜﻣ

،دﻮﺑ جاﺮﺨﺘــﺳا

ﺪﯿﻤﺳﻼﭘ ترﻮﺻ و ﺖﻓﺮﮔ ﺲﭙﺳ ﺎﺑ ﻢﻀﻫ ﯽــﻤﯾﺰﻧآ BamH1

و ﺪﯿﻤﺳﻼﭘXho1 ﺐﯿﮐﺮﺗﻮﻧ ﺪﺷ ﺪﯿﯾﺎﺗ ياﺮﺑ. ﻪﻧﺎﺴﻤﻫ يزﺎﺳ

نژ رد NA رﻮﺘﮐو ﯽﻧﺎﯿﺑ pET-21 ﻪﻌﻄﻗ ﯽﻧژ ﺎﺑ هدﺎﻔﺘﺳا زا

ﻢﯾﺰﻧآ ﺎﻫ ي هﺪﺷ ﻪﺘﻔﮔ زا

رﻮﺘﮐو ﻪﯿﻟوا اﺪــﺟ و دراو رﻮــﺘﮐو

،ﺪﺷ ﯽﻧﺎﯿﺑ ﻢﻀﻫ ﻦﯿﻨﭽﻤﻫ ﯽﻤﯾﺰﻧآ

رﻮﺘﮐو pET-21 مﺎــﺠﻧا ﺰﯿﻧ

ﯽﻤﯾﺰﻧآ ﻢﻀﻫ .ﺪﺷ pET21

و ﯽــﻧژ ﻪﻌﻄﻗ شﺮﺑ -NA

Tvector

ﻢﯾﺰﻧآ ﻂﺳﻮﺗ يﺎﻫ

BamH1 و ﺲﭙــﺳ .ﺖﻓﺮﮔ ترﻮﺻXho1

ﺺﯿﻠﺨﺗ رﻮﺘﮐو pET21 ﯽﻄﺧ هﺪﺷ مﺎﺠﻧا ﺺﯿــﻠﺨﺗ .ﺪــﺷ

نژ ﻪــﺑ NA شور Gel extraction ﺎــﺑ

ﺖــﯿﮐ QIA quick ترﻮﺻ

لﺎﺼﺗا و ﺖﻓﺮﮔ نژ

ﻪﺑNA رﻮﺘﮐو ﯽﻧﺎــﯿﺑ pET-21

ﯽﻄﺧ هرﺎﻤﺷ لوﺪﺟ ﻖﺒﻃ هﺪﺷ 5

ﺖﺒﺴﻧ ود رد ﯽﻟﻮــﻣ

1 ﻪــﺑ

1 و 1 ﻪﺑ 3 زا ﻪﻌﻄﻗ نژ فﺪﻫ ﻪﺑ رﻮﺘﮐو .ﺪﺷ مﺎﺠﻧا

ناﺰﯿﻣ:

داﻮﻣ درﻮﻣ زﺎﯿﻧ ياﺮﺑ لﺎﺼﺗا نژ ﺑﻪ NA

رﻮﺘﮐو

ﯽﻧﺎﯿﺑ

pET-21

ﯽﻄﺧ هﺪﺷ

داﻮﻣ 1

ﻪﺑ 1 1

ﻪﺑ 3

ي ﻪﻌﻄﻗ نژ)فﺪﻫ )( NA مﺮﮔﻮﻧﺎﻧ 5/ (

0 75

/0

رﻮﺘﮐو ﻪﻌﻄﻗ ) (pET21a) مﺮﮔﻮﻧﺎﻧ 5/ (

6 5/

6

ﺮﻓﺎﺑ 10X ) وﺮﮑﯿﻣ ﺮﺘﯿﻟ 1 (

1

T4DNA Ligase ﻢﯾﺰﻧآ

ﺮﺘﯿﻟوﺮﮑﯿﻣ) ( 1

1

(ﺮﺘﯿﻟوﺮﮑﯿﻣ)ﺮﻄﻘﻣ با

1 75

/0

(ﺮﺘﯿﻟوﺮﮑﯿﻣ)ﻞﮐ ﻢﺠﺣ

10 10

هﺎﮕﻧآ pET21-NA ﻪﺑ

نﺎــﺑﺰﯿﻣ ﯽﯾﺎــﯾﺮﺘﮐﺎﺑ TOP10f'

ﺪﻌﺘﺴﻣ يﺮﺘﮐﺎــﺑ و دﺮــﮐ اﺪــﯿﭘ لﺎﻘﺘﻧا هﺪﺷ يﺎــﻫ

مرﻮﻔــﺴﻧاﺮﺗ

،هﺪﺷ يور ﺖﯿﻠﭘ يﺎﻫ يوﺎﺣ ﯽﭙﻣآ و ﻦﯿﻠﯿــﺳ ﻦﯿﻠﮑﯾﺎــﺳاﺮﺘﺗ

ﺖﺸﮐ هداد

،ﺪﻧﺪﺷ ﺖﻬﺟ ﯽﯾﺎــﺳﺎﻨﺷ يﺎﻫﺪﯿﻤــﺳﻼﭘ ﺐــﯿﮐﺮﺗﻮﻧ

زا ﯽــﻨﻠﮐ يﺎــﻫ ﺪــﺷر هدﺮــﮐ ﺮــﺑ يور ﺖــﯿﻠﭘ يﺎــﻫ يوﺎــﺣ

ﯽﭙﻣآ ﯽﻧﻮﻠﮐ ،ﻦﯿﻠﯿﺳ وPCR

ﺖﺴﺗ rapid minipreb مﺎﺠﻧا

ﻞﺒﻗ ﺪﻨﻧﺎﻣ ﺲﭙﺳ .ﺪﯾدﺮﮔ زا

ﯽﻨﻠﮐ ﯽﯾﺎﻫ ﺞﯾﺎﺘﻧ ﻪﮐ نآ ﺎﻫ ﺖﺒﺜﻣ

،دﻮﺑ جاﺮﺨﺘﺳا مﺎﺠﻧا ﺪﯿﻤﺳﻼﭘ و ﺖــﻓﺮﮔ

ﺲﭙــﺳ ﺎــﺑ ﻢــﻀﻫ

ﯽﻤﯾﺰﻧآ BamH1 و ﺪﯿﻤﺳﻼﭘXho1 ﺐﯿﮐﺮﺗﻮﻧ ﺪﯿﯾﺎﺗ ﯽﯾﺎﻬﻧ

ﺪﺷ ﺪــﯾﺎﺑ ،ﺮﻈﻧدرﻮــﻣ ﻦﯿﺌﺗوﺮــﭘ ﺪﯿﻟﻮﺗ و نژ نﺎﯿﺑ ﯽﺳرﺮﺑ ﺖﻬﺟ.

ﺪﯿﻤﺳﻼﭘ ﯽﺗﻮﯾرﺎﮐوﺮﭘ نﺎﺑﺰﯿﻣ ﻞﺧاد ﻪﺑ ﺐﯿﮐﺮﺗﻮﻧ E. Coli

ﺳ ﯽﻧﺎﯿﺑ ﻪﯾﻮ ﻦﯾاﺮﺑﺎﻨﺑ ،ﺪﺑﺎﯾ لﺎﻘﺘﻧاBL21

ﯾ ﮏ ﮏﺗ نﻮﻠﮐ زا

يﺎﻫ ﯽﻨﻠﮐ دﺎﺠﯾا

هﺪﺷ رد ﻪﻠﺣﺮﻣ لﺎﻘﺘﻧا ﺪﯿﻤﺳﻼﭘ ﻪﺑ نﺎﺑﺰﯿﻣ

ﮏﯿﺗﻮﯾرﺎﮐوﺮﭘ ﺪﻌﺘﺴﻣ

ﻪﺑ هﺪﺷ ﻪﻠﯿﺳو كﻮﺷ ﯽﺗراﺮﺣ ار زا

ﺢﻄﺳ ﺖﯿﻠﭘ يﺎﻫ ﺖﺸﮐ ﻖﯿﻗر ﻪﺑ ﻪﻟﻮﻟ يﺎﻫ يوﺎﺣ 10 ﯽﻠﯿﻣ ﺮــﺘﯿﻟ

ﺖﺸﮐ ﻂﯿﺤﻣ ﻊﯾﺎﻣ

يوﺎﺣ ﯽﺘﻧآ ﮏﯿﺗﻮﯿﺑ ﯽﭙﻣآ ﻦﯿﻠﯿﺳ لﺎﻘﺘﻧا

ﯽﻣ ــﺷ نﻮﮑﻟﺎــﻓ .دﻮ ﺎــﻫ

رد رﻮﺗﺎــﺑﻮﮑﻧا رادﺮﮑﯿــﺷ

37 ﻪــﺟرد

ﯽﺘﻧﺎﺳ داﺮﮔ راﺮﻗ ﺪﻧﺮﯿﮔ ﯽﻣ ﺎﺗ

ﯽﻧﺎﻣز ﻪــﮐ ﻪــﺑ 600nm OD

ﻦﯿﺑ 4/

0 ﺎﺗ 6/

0 .ﺪﻨﺳﺮﺑ ﺲﭙﺳ 1 ﯽﻠﯿﻣ ﺮﺘﯿﻟ زا نآ ﻪــﺑ ناﻮــﻨﻋ

ﻪﻧﻮﻤﻧ ﺎﻘﻟاﺮﯿﻏ ﯽﻣ ﺖﺷادﺮﺑ و دﻮﺷ

ﻪﻨﯿﻬﺑ ﺖﻬﺟ يزﺎﺳ

يﺎــﻘﻟا

،نﺎﯿﺑ ﻞﯿﭘوﺮﭘوﺰﯾا ﺪﯾزﻮﺘﮐﻻﺎﮔﻮﯿﺗ

(IPTG) ﺎﺑ ﺖــﻈﻠﻏ يﺎــﻫ

،1mM 0.5mM و 0.1mM ﺖﻓﺮﮔ راﺮﻗ ﺶﯾﺎﻣزآ درﻮﻣ و

رد ﺖــﯾﺎﻬﻧ ياﺮــﺑ ﺮــﻫ ﻪــﻟﻮﻟ ﺎــﺑ ﺖــﻈﻠﻏ ﻒــﻠﺘﺨﻣ

،ﺮﮔﺎــﻘﻟا

(5)

195 ﻦﯾدروﺮﻓ ، 1400

بﻮﯿﺗوﺮﮑﯿﻣ ﯽﯾﺎﻫ

يوﺎﺣ يﺮﺘﮐﺎﺑ بﻮﺳر زا

نﺎﻣز 0 ﺎﺗ 5 و

ــﯾ ﮏ ﻪــﻧﻮﻤﻧ 16 ﻪﺘﻋﺎــﺳ ) Over night ﻢﯿﻫاﻮــﺧ (

.ﺖــﺷاد

ﻪﻧﻮﻤﻧ ﺲﭙﺳ ﺎﻫ

يور لژ ود ﻢﯾﺪــﺳ زا و هدﺮــﺑ ﺪﯿﻣآ ﻞﯾﺮﮐا

تﺎﻔﻟﻮﺳ ﻞﯿﺳد (SDS-PAGE)

12 هدﺎﻔﺘــﺳا ﺪﺻرد ﺪــﻌﺑ ﺪــﺷ

زا نﻮﯿﺳاﺰﯿﻟﺎﻣﺮﻧ و

هدﺎﻣآ

،يزﺎﺳ 15 ﺮﺘﯿﻟوﺮﮑﯿﻣ زا

ﺮﻫ ﻪﻧﻮﻤﻧ

ﺖﻬﺟ ﺪــﯿﯾﺎﺗ نﺎــﯿﺑ ﻦﯿﺌﺗوﺮــﭘ درﻮــﻣ ﺮــﻈﻧ رد SDS-PAGE

هدﺎﻔﺘﺳا هﺪﺷ ﺖﺳا و ﺪــﯿﯾﺎﺗ ﺖــﻬﺟ تﻼﺑ نﺮﺘﺳو ﻦﯿﺌﺗوﺮــﭘ

ﺮﻈﻧﺪﻣ ﻪــﮐ ياراد مد ﯽﻨﯾﺪﯿﺘــﺴﯿﻫ ﯽــﻠﭘ ﺖــﺳا

ﺎــﺑ هدﺎﻔﺘــﺳا

ﯽﺘﻧآزا ﺎــﺑ ﻪــﮔوﮋﻨﮐ ﺲﯿﻫ ﺎــﺑHRP

ﺖــﻗر 6000 /1 ﻪــﯿﻠﻋ مد

ﯽﻠﭘ ﺪﯿﺘﺴﯿﻫ ﯽﻨﯾ و ﻦﯿﺌﺗوﺮﭘ ﺎﺑ ﻞﯿﺘﻣاﺮﺘﺗ ياﺮﺘﺴﺑﻮﺳ زا هدﺎﻔﺘﺳا

ﻦﯾﺪﯾﺰﻨﺑ ) ) ﺖﮐﺮــﺷ زا(TMB 110M1427

( SIGMA ا مﺎــﺠﻧ

.ﺪــﺷ ﺮــﻈﻧ درﻮــﻣ ﺮﮐرﺎــﻣ Prestained Protein Ladder

ﺳ ﺖﮐﺮﺷ ﯿ نﻮﻠﮐﺎﻨ دﻮﺑ .

و ﺎﻫ ﻪﺘﻓﺎﯾ ﺚﺤﺑ

لﺎﺳ ﯽﻃ ﺮﺿﺎﺣ ﺶﻫوﮋﭘ يﺎﻫ

1399 - 1398 ﺶﺨﺑ رد

اﺰﻧاﻮﻠﻔﻧآ ي د .ﺖﺳا هﺪﺷ مﺎﺠﻧا ناﺮﻬﺗ رﻮﺘﺳﺎﭘﻮﺘﯿﺘﺴﯿﻧا ر

ﺮﯾﻮــﺼﺗ

هرﺎﻤﺷ 1 ﺑ ﺪﻧﺎﺑ ﻪ ﺖﺳد هﺪﻣآ زا زرﻮــﻓوﺮﺘﮑﻟا ﻂــﺳﻮﺗ لﻮــﺼﺤﻣ

ﻞﻣﺎﺷ ﻪﮐPCR نژ ﻪﺑ NA هاﺮﻤﻫ هﺎﮕﯾﺎﺟ يﺎﻫ شﺮﺑ ود ﻢﯾﺰﻧآ

BamH1/Xho1 ﻪﮐ

ﻂﺳﻮﺗ ﺎﻫﺮﻤﯾاﺮﭘ ﯽﺣاﺮﻃ ،ﺖــﺳا هﺪﺷ

ﯽﻣ نﺎﯾﺎﻤﻧ ﯽﺑﻮﺧ ﻪﺑ هزاﺪﻧا .ﺪﺷﺎﺑ

ﻦﯾا ﯽﻟاﻮﺗ 975bp .ﺖﺳا

3 2 1

ﺮﯾﻮﺼﺗ ﺷ هرﺎﻤ

زرﺎﮔا لژ يور زرﻮﻓوﺮﺘﮑﻟا1:

1 لﻮﺼﺤﻣزا ﺪﺻرد

PCR

نژ ياﺮﺑ .NA

ﮏﻫﺎﭼ 1 -

1 kb DNA ladder (Cinnagen Cat.NO.PR901644)

،

ﮏﻫﺎﭼ 2 - لﺮﺘﻨﮐ ﯽﻔﻨﻣ مﺎﺠﻧا)

نوﺪﺑPCR

ﻮﮕﻟا

،(

ﮏﻫﺎﭼ 3 - ﺶﻨﮐاو ﻪﻌﻄﻗ ياﺮﺑPCR

هزاﺪﻧا ﺎﺑNA

975bp

ﻣ ﯽ ﺪﺷﺎﺑ .

رد ﺮﯾﻮﺼﺗ هرﺎﻤﺷ 2 ﻢــﻀﻫ زا ﻞــﺻﺎﺣ ﺞﯾﺎﺘﻧ ﯽــﻤﯾﺰﻧآ

ﻼﭘ يﺎﻫﺪﯿﻤﺳ ﺐﯿﮐﺮﺗﻮﻧ

pET21-NA ﻪﺑ

ﻪﻠﯿﺳو ﻢﯾﺰﻧآ يﺎــﻫ

BamH1 و ﯽﻣ هﺪﻫﺎﺸﻣ ارXho1 رد ﻪﮐ ﺪﯿﻨﮐ

ﺮﯾﻮﺼﺗ جوﺮــﺧ

ﻪﻌﻄﻗ 975bp نﺎﺸﻧ ﻪﻧﺎﺴﻤﻫ ﺖﺤﺻ هﺪﻨﻫد يزﺎﺳ

ﯽــﻣ .ﺪــﺷﺎﺑ

ﺮﯾﻮﺼﺗ هرﺎﻤﺷ 3 ﻪﺸﻘﻧ pET21 ﻪﺑ ﺶﻫوﮋــﭘ ﻦﯾارد ﻪﺘﻓر رﺎﮐ

ﯽﻣ نﺎﺸﻧ ار .ﺪﻫد

ﺮﯾﻮــﺼﺗ هرﺎﻤــﺷ

زرﻮــﻓوﺮﺘﮑﻟا2:

طﻮــﺑﺮﻣ ﻪــﺑ ﻪــﺠﯿﺘﻧ ﻢــﻀﻫ ﯽــﻤﯾﺰﻧآ

PET21-NA

يور لژ زرﺎﮔآ 1 ﺪﺻرد ﮏﻫﺎﭼ 1 -

1 kb DNA ladder

(Cinnagen Cat.NO. PR901644)

و ﮏﻫﺎﭼ 2 - لﻮــﺼﺤﻣ ﻢــﻀﻫ ﺎــﺑ

ود ﻢﯾﺰـــﻧآ

BamH1

و وXho1

جوﺮـــﺧ ﻪـــﻌﻄﻗ 975bp

نژ وNA

ﻪﻌﻄﻗ ي 5443bp

رﻮﺘﮐو

PET21

ﺪﯿﯾﺎﺗ زا ﻞﺻﺎﺣ ﺞﯾﺎﺘﻧ نﺎــﯿﺑ

ﻦﯿﺌﺗوﺮــﭘ ﻂــﺳﻮﺗﺮﻈﻧدرﻮﻣ

SDS-PAGE رد

ﺮﯾﻮﺼﺗ هرﺎﻤــﺷ 4 ﺎــﺑ ،ﺖــﺳا هﺪــﺷ ﻪــﺋارا

ﻪــﺟﻮﺗ ﻪــﺑ ﻦــﯾا ﻪــﮐ ﯽــﺒﯾﺮﻘﺗ نزو ﻦﯿﺌﺗوﺮــﭘ

ﺐـــﯿﮐﺮﺗﻮﻧ

زاﺪﯿﻨﯿﻣآرﻮﻧ ادوﺪﺣ

38

،ﺖﺳا نﻮﺘﻟادﻮﻠﯿﮐ ﺪــﻧﺎﺑ

ﯽــﺼﺨﺸﻣ

رد نآ ﻪــﯿﺣﺎﻧ زا لژ هﺪﻫﺎــﺸﻣ و ﺪــﺷ زا ﻞــﺻﺎﺣ ﻦﯿﺌﺗوﺮــﭘ

ﺖﻈﻠﻏ 5/

0 ﯽﻠﯿﻣ زا زﻻﻮﻣ نﺎــﻣز تﺪﻣ وIPTG

16 ﺖﻋﺎــﺳ

وﺰــﻓا زاﺪﻌﺑ ــﺘﻬﺑ ناﻮــﻨﻋ ﻪــﺑ ،ﺮﮔﺎــﻘﻟا ند

ﻦﯿﺌﺗوﺮــﭘ نﺎــﯿﺑ ﻦﯾﺮ

.ﺪﯾدﺮﮔ بﺎﺨﺘﻧا ﻪﻃﻮﺑﺮﻣ

رد ﺮﯾﻮﺼﺗ هرﺎﻤﺷ 5 ﻞﺻﺎﺣ ﺞﯾﺎﺘﻧ زا

نﺮﺘﺳو تﻼﺑ ﺎــﺑ

هدﺎﻔﺘﺳا زا يدﺎــﺑ ﯽــﺘﻧآ ﻪــﯿﻠﻋ

ﯽﻟاﻮــﺗ His ﻪــﮐ رد ياﺪــﺘﺑا

ﯽﻟاﻮﺗ ﻪﮐﺪﯿﯾﺎﻤﻧ ﯽﻣ ﻪﻈﺣﻼﻣ ار ﺖﺳا هﺪﺷ نژﻮﯿﻓNA

ﺪﻧﺎﺑ

طﻮــﺑﺮﻣ ﻪــﺑ ﻦﯿﺌﺗوﺮــﭘ NA ﺎــﺑ رﺎــﻈﺘﻧا درﻮــﻣ نزو 38KD

ﺖﺳا نﺎﯾﺎﻤﻧ .

975bp 5443

bp 975 bp

2

(6)

ﺮﯾﻮﺼﺗ رﺎﻤﺷ ه

ﯽﻧﺎﯿﺑرﻮﺘﮐو ﻪﺸﻘﻧ 3:

pET21

ﺮﯾﻮﺼﺗ هرﺎﻤﺷ 4 : SDS-PAGE ﻪﻧﻮﻤﻧ

يﺎﻫ بﻮﺳر يﺮﺘﮐﺎﺑ BL21 ﺶﯿــﭘ و

ﺲﭘ زا يﺎﻘﻟا نﺎــﯿﺑ ﻦﯿﺌﺗوﺮــﭘ ﺐــﯿﮐﺮﺗﻮﻧ ﺎــﺑ NA ﺖــﻈﻠﻏ 5/

0 ﯽــﻠﯿﻣ رﻻﻮــﻣ ﺮﮔﺎــﻘﻟا

ردIPTG يﺎﻣد 37 °C نﻮﺘــﺳ ، 1 - ﻪــﻧﻮﻤﻧ ﻦﯿﺌﺗوﺮــﭘ ﺶﯿــﭘ زا نﻮﺘــﺳ ،ﺎــﻘﻟا 2 ﺎــﺗ 6 -

يﺎﻫ ﻪﻧﻮﻤﻧ ﯽﻨﯿﺌﺗوﺮﭘ تﺎﻋﺎﺳ 1 ﺎﺗ 5 ﺲﭘ زا ﺎﻘﻟا نﻮﺘــﺳ ،نﺎــﯿﺑ 7 - ﻪــﻧﻮﻤﻧ ي ﯽــﻨﯿﺌﺗوﺮﭘ

16 )ﺖﻋﺎﺳ Over night ( ﺲﭘ زا ﺎﻘﻟا نﺎﯿﺑ .ﺪﺷﺎﺑ ﯽﻣ نزو ﯽﻟﻮــﮑﻟﻮﻣ ﻦﯿﺌﺗوﺮــﭘ

ﺐﯿﮐﺮﺗﻮﻧ ﺮﻈﻧدرﻮﻣ 38 نﻮﺘﻟادﻮﻠﯿﮐ ﺖﺳا

.

ﺮﮐرﺎﻣ- 1.ﺖﺳازاﺪﯿﻨﯿﻣآرﻮﻧ ﺐﯿﮐﺮﺗﻮﻧ ﻦﯿﺌﺗوﺮﭘ تﻼﺑ نﺮﺘﺳو:5هرﺎﻤﺷﺮﯾﻮﺼﺗ ،نﻮﻠﮐﺎﻨﯿﺳ ﺖﮐﺮﺷ زا هﺪﺷ ﻪﯿﻬﺗ ﯽﻟﻮﮑﻟﻮﻣ نزو 2

- ﻨﻣ لﺮﺘﻨﮐ ﯽﻔ ) ﺪﻗﺎﻓ يﺮﺘﮐﺎﺑ

،(ﯽﻧﺎﯿﺑ ﺪﯿﻤﺳﻼﭘ 3

- ﺐﯿﮐﺮﺗﻮﻧ ﻦﯿﯿﺗوﺮﭘ يوﺎﺣ ﻪﻧﻮﻤﻧ NA

38KD

(7)

195 ﻦﯾدروﺮﻓ ، 1400

رد لﺎــﺳ 2020 ﺎــﺑ ﺶﻧارﺎــﮑﻤﻫ و ﯽﻬﻠﻟاﺪــﺒﻋ يدﻼﯿــﻣ

ﯽــﺳرﺮﺑ 28 سوﺮــﯾو مﻮــﻧژ عﻮــﻧ شدﺮــﮔ لﺎــﺣردH5NX

سوﺮﯾو ﻦﯾا رﺎﺸﺘﻧا زا يﺪﻫاﻮﺷ ﻪﮐ ﺪﻨﺘﺷاد نﺎﻋذا ،ناﺮﯾارد ﻦــﮑﻤﻣ ناﺮــﯾا ﻦﯾاﺮﺑﺎــﻨﺑ ،دراد دﻮﺟو ﻪﻧﺎﯿﻣروﺎﺧ ﻪﺑ ناﺮﯾا زا ﺖــﺳا دﺎــﺣ قﻮــﻓ سوﺮــﯾو رﺎــﺸﺘﻧا ياﺮــﺑ غاد ﻪــﻄﻘﻧ ﮏــﯾ

عﻮﻨﺗ دﺎﺠﯾا و نﺎﮔﺪﻧﺮﭘ ﺪﯾﺪﺟ ﯽﮑﯿﺘﻧژ

ﺪــﺷﺎﺑ )5 نژ .( درﻮــﻣ

ﻪـــﻌﻟﺎﻄﻣ ﻦـــﯾا رد ﯽـــﺳرﺮﺑ نژ

ﻪـــﮐ دﻮـــﺑ سوﺮـــﯾوNA

ﯽﺘﻧآ نژ ردﺎﻗ نآ يرﺎﺘﺧﺎﺳ يﺎﻫ لارﻮــﻣﻮﻫ ﯽــﻨﻤﯾا ﺦﺳﺎﭘ ﺖﺳا

ار ﺪﻨﮐ ﮏﯾﺮﺤﺗ )

10،7،1 نﺎﻘﻘﺤﻣ .( ﺪــﻧروﺎﺑ ﻦــﯾا ﺮﺑ يرﺎﯿﺴﺑ

شور ﻪــﮐ اﺰﻧاﻮــﻠﻔﻧآ ﯽــﺼﯿﺨﺸﺗ يﺎــﻫ )ﯽــﻏﺮﻣ ي

(H5N1

ﯽﺘﻧآ ﻪﯾﺎﭘﺮﺑ يدﺎــﺑ

رﺎﯿــﺴﺑ لﺎــﻧﻮﻠﮐ ﯽــﻠﭘو لﺎــﻧﻮﻠﮐﻮﻧﻮﻣ يﺎــﻫ

شور تﻼﮑﺸﻣ و هدﻮﺑ ﺮﺗﺪﯿﻔﻣ شور ﻞــﯿﺒﻗ زا ﯽﺘﻨــﺳ يﺎﻫ

) نﻮﯿــﺳﺎﻨﯿﺗﻮﻠﮔﺎﻤﻫ رﺎــﻬﻣ شور و (HI

ﺲﻧﺎــﺳرﻮﻠﻓﻮﻧﻮﻤﯾا يﺎــﻫ

ﺪــﻧراﺪﻧ ار ) 12،11 . ( ﻦــﺴﮐاو ﻪــﮐ ﺪــﻨﺘﻓﺎﯾرد نﺎــﻘﻘﺤﻣ يﺎــﻫ

نژ ﯽــﺘﻧآ ﻪﯾﺎﭘﺮﺑ اﺰﻧاﻮﻠﻔﻧآ ﯽــﻣ ،سوﺮــﯾوNA

رﺎﯿــﺴﺑ ﺪــﻧاﻮﺗ

ﻦﺴﮐاو ﺪﯾﺎﺑ ﻪﮐ ﺪﻧراد هﺪﯿﻘﻋ و هدﻮﺑ ﺪﯿﻔﻣ ﺎــﺑ ار ﯽــﻠﻌﻓ يﺎﻫ

و ﺪﻨﺸﺨﺒﺑ دﻮﺒﻬﺑNA ﻦــﺴﮐاو ﮏﯾ ناﻮﻨﻋ ﻪﺑ نآ زا ﺪﯾﺎﺑ ﺎﯾ

ياﺮﺑ هﻮﻘﻟﺎﺑ ﻦــﺴﮐاو ﯽﻨﯾﺰﮕﯾﺎــﺟ

ياﺮــﺑ ﯽــﻠﻌﻓ لﺎــﻌﻓﺮﯿﻏ يﺎــﻫ

زوﺮﺑ زا يﺮﯿﮔﻮﻠﺟ دﺮﮐ هدﺎﻔﺘﺳاAI

) 17 - 13 ﻪﺑ .( لﺎﺒﻧد ﻪــﯾﺰﺠﺗ و

ﻞــﯿﻠﺤﺗ يﺎــﻫ ﯽﮑﯿﺗﺎــﻣرﻮﻔﻧاﻮﯿﺑ ﻪــﯿﻟوا

نژ يور زاNA

ﺮــﻈﻧ

ﺺﺧﺎﺷ يﺎﻫ

،ﻒﻠﺘﺨﻣ ﻢﯾدﺮﮐ هﺪﻫﺎﺸﻣ ﻪــﮐ

لﻮــﮑﻟﻮﻣ NA

ﯽﻣ ﺪﻧاﻮﺗ ﻪﺑ ﯽﺑﻮﺧ و ﻪــﺑ ترﻮــﺻ راﺪــﯾﺎﭘ رد ﮏــﯾ نﺎــﺑﺰﯿﻣ

ﮏﯿﺗﻮﯾرﺎﮐوﺮﭘ نﺎﯿﺑ

دﻮﺷ و ﺎﮑﻣ سﺎﺳاﺮﺑ ن

يﺎﻫ ﯽــﭘا ﯽﭘﻮــﺗ

ﺶﯿﭘ ﯽﻨﯿﺑ

،هﺪﺷ ﻪﺑ رﻮﻃ هﻮﻘﻟﺎﺑ ردﺎﻗ ﻪﺑ ﮏﯾﺮﺤﺗ ﻢﺘــﺴﯿﺳ ﯽــﻨﻤﯾا

نﺪﺑ ﻪﯿﻠﻋ سوﺮﯾو ﺖــﺳا ﯽــﻣ و ﻪــﺑ ﺪــﻧاﻮﺗ رد ﻢﯿﻘﺘــﺴﻣ رﻮــﻃ

يژﻮﻟوﺮﺳ تﺎﺸﯾﺎﻣزآ دﻮﺷ هدﺎﻔﺘﺳا ﺺﯿﺨﺸﺗ ياﺮﺑ

ﻞﯾﻻد.

هدﺎﻔﺘﺳا زا رﻮﺘﮐوTA ﻞﺒﻗ زا هدﺎﻔﺘﺳا زا رﻮﺘﮐو ﯽﻠﺻا ﻦﯾا

ﺖﺳا ﻪﮐ ﮓﻨﯿﻧﻮﻠﮐ ﺎﺑ ﻦﯾا شور ﻪﺑ ﺖﻋﺮﺳ ﯽﻣ مﺎﺠﻧا دﻮــﺷ و

ﯽﻟاﻮﺗ ﺮﻈﻧدرﻮﻣ ﻪﺑ ﻞﯿﻟد ﻦﺘﺷاد A Overhang رد

يﺎﻬﺘﻧا

دﻮﺧ ﻪﮐ ﻂﺳﻮﺗ ﻢﯾﺰﻧآ Taq polymerase دﺎﺠﯾا

ﯽــﻣ دﻮــﺷ

ﻪﺑ يﺎﻫﺪﯿﺗﻮﺌﻠﮐﻮﻧ ردT

رﻮﺘﮐو ﺘﻣ ﻞﺼ هﺪﺷ و ﮓﻨﯿﻧﻮﻠﮐ ﻪــﺑ

ﯽﺘﺣار مﺎﺠﻧا ﯽﻣ ﻢﺘــﺴﯿﺳ .دﻮــﺷ ﯽــﮑﯾpET

زا يﻮــﻗ ﻦﯾﺮــﺗ

ﻢﺘﺴﯿﺳ يﺎﻫ ﻪﺘﺧﺎﻨﺷ هﺪﺷ ياﺮﺑ ﮓﻨﯿﻧﻮﻠﮐ و نﺎﯿﺑ ﻦﯿﺌﺗوﺮﭘ يﺎــﻫ

ﯽﻠﮐﺎﯿﺷﺮﺷا يﺮﺘﮐﺎﺑرد ﺐﯿﮐﺮﺗﻮﻧ ﯽﻣ

ﻦﯾارد ﻦﯾاﺮﺑﺎﻨﺑ ،ﺪﺷﺎﺑ

ﯽﺗﻮﯾرﺎــﮐوﺮﭘ ﻢﺘــﺴﯿﺳ زا ﺎﻣ ﻪﻌﻟﺎﻄﻣ pET21

نﻮــﻠﮐ ﺖــﻬﺟ

نژ ﻢﯾدﺮﮐ هدﺎﻔﺘﺳاNA )6

نآزا .( ﯽﯾﺎﺟ ﺘﮐﺎﺑ ﻪﮐ يﺮ نﺎــﺑﺰﯿﻣ

ياﺮﺑ ﻦﯾا رﻮــﺘﮐو يﺮﺘﮐﺎــﺑ E.coli ﯽــﺳﺪﻨﻬﻣ هﺪــﺷ ﺎــﺑ مﺎــﻧ

،ﺖﺳاBL21 ﺲﭘ ﻦﯾاﺮﺑﺎﻨﺑ ﺮﯿﺜﮑﺗ زا

رد ﻪﯾﻮﺳ TOP10f' ﻪﺑ

ﻪﯾﻮﺳ ﯽﻧﺎﯿﺑ ﻪﺑBL21 ﯽﺑﻮﺧ ﻞﻘﺘﻨﻣ ﺪﺷ و نﺎﯿﺑ ﻞــﺑﺎﻗ ﻪــﺟﻮﺗ

ﻦﯿﺌﺗوﺮﭘ رد نﺎﻣز 16 و ﺖﻋﺎــﺳ ناﺰــﯿﻣ

5 /0 ﯽــﻠﯿﻣ رﻻﻮــﻣ زا

ﺮﮔﺎﻘﻟا ﺐﺳﺎﻨﻣIPTG

ندﻮﺑ ﻦﯾا ﻢﺘﺴﯿﺳ ﯽﻧﺎــﯿﺑ ار ياﺮــﺑ ﻟﻮﺗ ﺪــﯿ

ﺐﯿﮐﺮﺗﻮﻧ ﻦﯾا ﺪﯿﯾﺎﺗ ﻦﯿﺌﺗوﺮﭘ ﺎﺑ .ﺪﻨﮐ ﯽﻣ

ﺞﯾﺎﺘﻧ ﻪﺑ ﻪﺟﻮﺗ ﻪــﺑ

ﺖﺳد هﺪﻣآ زا SDS-PAGE ﺎﻫﺪــﻧﺎﺑ ﻦﯿﺌﺗوﺮــﭘ ﺖﻈﻠﻏ ﺰﯿﻟﺎﻧآو

ﻂﺳﻮﺗ Image ﺑ ، ﻦﯾﺮــﺘﻬ نﺎــﯿﺑ ﺖــﻈﻠﻏ رد 5

/0 ﯽــﻠﯿﻣ رﻻﻮــﻣ

و نﺎﻣز تﺪﻣرد ) زور ﻪﻧﺎﺒــﺷ ﮏﯾ

16 (ﺖﻋﺎــﺳ ﺲــﭘ زا ﺎــﻘﻟا

بﺎﺨﺘﻧا ﺖﻬﺟو ﺪﺷ ﺪﯿﯾﺎﺗ

ﻦﯿﺌﺗوﺮﭘ ﺐﯿﮐﺮﺗﻮﻧ ياراد ﻪــﻟﺎﺒﻧد

ﻨﯾﺪﯿﺘﺴﯿﻫ نﺎﯿﺑ ﯽ هﺪﺷ زا شور نﺮﺘﺳو تﻼﺑ هدﺎﻔﺘﺳا .ﺪﺷ

يراﺰﮕﺳﺎﭙﺳ

ﻦﯾا ﻪﻟﺎﻘﻣ ﻞﺻﺎﺣ نﺎﯾﺎﭘ ﺎﺑ ﻪﻣﺎﻧ ناﻮﻨﻋ

"

يزﺎﺳ ﻪﻧﺎﺴﻤﻫ

اﺰﻧاﻮﻠﻔﻧآ سوﺮﯾو زاﺪﯿﻨﯿﻣآرﻮﻧ ﺐﯿﮐﺮﺗﻮﻧ نژ ﯽﺘﻧآ نﺎﯿﺑ و ﻪﺑA

ﺖﺴﺗ رد هدﺎﻔﺘﺳا رﻮﻈﻨﻣ يﺎﻫ

ﯽﺼﯿﺨﺸﺗ

"

ﻊﻄﻘﻣ ﯽﺳﺎﻨﺷرﺎﮐ

ﺮﺘﺷا ﮏﻟﺎﻣ هﺎﮕﺸﻧاد ﺪﺷرا ﯽﻣ

ﺪﺷﺎﺑ ﻪﮐ هژوﺮﭘ مﺎﻤﺗ رد

ﺶــﺨﺑ

اﺰﻧاﻮﻠﻔﻧآ و ﺮﯾﺎﺳ سوﺮﯾو يﺎﻫ ﯽﺴﻔﻨﺗ ﻮﺘﯿﺘﺴﻧا رﻮﺘــﺳﺎﭘ ناﺮــﯾا و

ﺖﯾﺎﻤﺣ ﺎﺑ قﻼﺧاﺪــﮐ ﺎــﺑ نآ ﯽﻟﺎﻣ IR.pll.REC.1395.82

.ﺪﺷ مﺎﺠﻧا

References

1. Najafi S, Behzadian F, Fotuhi F, Fallah Mehrabadi J. Construction of a recombinant bacmid DNA in order to express Neuraminidase gene of influenza virus H1N1. J Arak Univ Med Sci 2012; 15(5): 58-65 (Persian).

2. Farahmand B, Khodabandeh M, Mahboudi F, Fotouhi F, Barkhordari F, Saleh M, et al.

Isolation, cloning, and sequencing of influenza A (H1N1) hemagglutinin for production of hemagglutinin gene bank. J Arak Univ Med

(8)

Sci 2011; 13(53): 59-67 (Persian).

3. Behzadian F, Goodarzi Z, Saberfar E.

Construction of a new recombinant baculovirus encoding HA, NA, and M1 proteins of swine influenza (H1N1) virus and its expression in insect cells. J Arak Univ Med Sci 2013;

15(8): 16-25 (Persian).

4. Aliakbari M, Behzadian F, Deldar AA, Mahyad B, Bidram M, Bahreinian M. Secretory expression of hemagglutinin globular domain (HA1) of the influenza A (H5N1) virus in Bacillus subtilis. Iran J Med Microbiol 2015;

9(3): 48-53 (Persian).

5. Abdollahi H, Ali ASM, Monne I, Milani A, Habibi M, Zamperin G, et al. Spatial spread and emergence of reassortant H5 highly pathogenic avian influenza viruses in Iran reassortant H5 highly pathogenic avian influenza virus in Iran. Infect Genet Evol 2020; 83: 104342.

6. Fiebig L, Soyka J, Buda S, Buchholz U, Dehnert M, Haas W. Avian influenzaA (H5N1) in humans: new insights from a line list of World Health OrganizaIon confirmedcases, September 2006 to August 2010. Euro Surveillance: BulleIn Europeen Sur Lesmaladies Transmissibles=European Communicable Disease BulleIn 2011; 16(32).

7. Subathra M, Santhakumar P, Satyam Naidu S, Lakshmi Narasu M, Senthilkumar TMA, Lal SK. Expression of Avian Influenza Virus (H5N1) Hemagglutinin and Matrix Protein 1 in Pichia pastoris and Evaluation of their Immunogenicity in Mice. Appl Biochem Biotechnol 2014; 172: 3635-3645.

8. Vincze T, Posfai J, Roberts RJ. NEBcutter: A program to cleave DNA with restriction enzymes. Nucleic Acids Res 2003; 31(13):

3688-3691.

9. NEBcutter V2.0. 2020. Available from:

http://www.labtools.us/nebcutter-v2-0.

10. Behzadian, F, Goodarzi Z, Fotouhi F, Saberfar E. Baculoviral Co-Expression of HA, NA and M1 Proteins of Highly Pathogenic H5N1 Influenza Virus in Insect Cells. Jundishapur Journal of Microbiology 2013; 6(9): e7665 (Persian).

11. Shoman SA, Khodier MH. Rapid detection of the avian influenza virus H5N1 subtype in Egypt. African Journal of Biotechnology 2013; 12(19).

12. Khurana S, Sasono P, Fox A, Van Kinh N, Thai PQ, Hien NT, Liem NT, Horby P, Golding H. H5N1-SeroDetect EIA and rapid test: a novel differential diagnostic assay for serodiagnosis of H5N1 infections and surveillance. Journal of Virology 2011;

85(23): 12455-12463.

13. Eichelberger MC, Monto AS. Neuraminidase, the forgotten surface antigen, emerges as an influenza vaccine target for broadened protection.

The Journal of Infectious Diseases 2019;

219(Supplement_1): S75-S80.

14. Chen P, Ding L, Jiang Y, Zeng X, Deng G, Shi J, Li Y, Liu L, Zhao Y, Hu Y, Liu J.

Protective efficacy in farmed ducks of a duck enteritis virus-vectored vaccine against H5N1, H5N6, and H5N8 avian influenza viruses.

Vaccine 2019; 37(40): 5925-5929.

15. Schön J, Ran W, Gorka M, Schwemmle M, Beer M, Hoffmann D. A modified live bat influenza A virus-based vaccine prototype provides full protection against HPAIV H5N1. Npj Vaccines 2020; 5(1): 1-12.

16. Gomaa MR, Khalil AA, Kandeil A, Sabir JS, Kayed A, Moatasim Y, El-safty MM, Kayali G, Ali, MA. Development of an effective contemporary trivalent avian

(9)

195 ﻦﯾدروﺮﻓ ، 1400

influenza vaccine against circulating H5N1, H5N8, and H9N2 in Egypt. Poultry science 2019; 98(12): 6289-6295.

17. Giurgea LT, Morens DM, Taubenberger JK,

Memoli MJ. Influenza Neuraminidase: A Neglected Protein and Its Potential for a Better Influenza Vaccine. Vaccines 2020;

8(3): 409.