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—BoCaca— BAO CAO SAN P H A M NGHIEN CUtJ DE TAI CAP BO TSn de tai: NGHIEN c i r u ifNG DVNG QUI TRINH SAN XUAT KHANG HUYET THANH Sdl, BAI LIET, ROTA SLT DVNG KIEM DINH VAC XIN Chu nh

O'IZZ-I !

2010

^

BOYTli

BAO CAO SAK PHAM NGIHtN ClJlJ » i TM CiP Bd

T«nd^t^i:

NGHlfiN CCti tJMG DUNG Qtl TRINH sAiN XUIT KIt4NG HUYIT TH4NH

sen, B ^ LifiT, ROTA SlJ DIING KJfiM B.INH v i c XIN

Citii n h l ^ S4 Ui: PGS TS U TW Luto

Cor quan tj6 chite) cM tri dS tM:

TruBg tdm NgWto «hi Sia xaa'f Vfc xin vh Sinh ph&i Y t«f

Mas6Mm{aine&y

Niton-2010

—BoCaca—

BAO CAO SAN P H A M NGHIEN CUtJ DE TAI CAP BO

TSn de tai:

NGHIEN c i r u ifNG DVNG QUI TRINH SAN XUAT KHANG HUYET THANH

Sdl, BAI LIET, ROTA SLT DVNG KIEM DINH VAC XIN

Chu nhiem de tai: PGS.TS.LI Thi LuSn

Off quan (Th cbvc) chii tri dl tai:

Trung tam Nghien ciru San xuat Vac xin va Sinh pham Y te

Ma so dl tki (neu co):

Nam 2010

sofi3ca

BAO CAO SAN PHAM NGHIEN ciTu DE TAI CAP BO

Ten de tai:

NGHIEN CCtJ ITNG DVNG QUI TRINH SAN XUAT KHANG HUYET THANH sen, B^I LIIPT, ROTA SU^ DyNG KIEM DINH VAC XIN

Chii nhifm de tai:

Co* quan chu tri de tai:

Cap quan ly:

Ma s^ dk tki (n^u co):

Then gian thu-c hi^n:

Tong kinh phi thifc hi^n de tai:

Trong d6: kinh phi SNKH :

Ngucin kh^c (neu c6):

PGS.TS.Le Thj Luan Trung tSm Nghien cihi San xuat Vac xin va Sinh pham Y te

BOYte

tir thang 7 nam 2007 den thang 12 nam 2009

910 trifu dong

598 tri^u dong tri^u dong

Nam 2010

1 T£n de tai:

NGHIEN c u t ) iTNG DyNG QUI TRINH SAN XUAT KHANG HUYET THANH SOI, BAI LIET, ROTA SU^ DUNG KIEM DINH VAC XIN

2 Chu nhifm dl tai: PGS.TS.Le Thi Luan

3 Co quan chu tri de tai:

Trung tam Nghien cuu San xudt Vic xin va Sinh phAm Y t^

4 Cff quan quan ly de tai: Bg Y t^

5 Thir ky detail

6 Pho chu nhifm de tai ho|lc ban chii nhifm de tai (neu co):

7 Danh sach nhirng ngiroi thirc hifn chinh:

Nguyen Dang Hien

Nguyen Thi Quy

Le Trung Dung

Nguyen Thiiy Huang

Nguyen Thi Mai Huong

Tran Bich Hanh

Dang Mai Dung

Nguyen Nu Anh Thu

Ngo Thu Huang

8 C^c de t&i nhanh (de muc) ciia de tai (neu co)

9 Thoi gian thyc hien de tai: tu thang 7 nam 2007 d6n thang 12 nam 2009

Danh muc san pham

1 Qui trinh cong nghf san xuat huyet thanh soi

2 Qui trinh cong nghe san xuat huyet thanh bai lift

3 Qui trinh cong nghf san xuat huyet thanh Rota

4 Tieu chuan ca stf huyet thanh soi

5 Tieu chuan ca sa huyet thanh b^i lift

6 Tieu chuan ca so* huyet thanh Rota

7 Bai bao da dang

8 Dao t^o Thac sy

I Qui trinh san xuat huyet thanh sdi

Nguyen vft lieu

Khang nguyen Soi tit chung Edmonston do vien Kitasato cung cap;

Tho trong lugng ttr 1,8 ~ 2,5 kg;

Te bao Veto do vien Kitasato-Nhat ban cung cip;

Moi trucmg hoa ch4t: MEM, NaHCOs 7,5%, BS, PBS (-), khang sinh P-S, Hank, chit bao quan vacxin, ta dugc tiem dap ling mien dich (Adjuvant): Ta dugc hoan chinh (CFA) va ta dugc khong hoan chinh(IFA)

Vat lieu : Bom kim tiem trgn ta dugc, tu im CO2, pipet Aid, pipetman, diu tip cac loai, may ly tam lanh, ly tam thucmg, chai chiia mau (100 ml, 500 ml ), phiSn 24 giSng Dung

cu lay mau tho,

Phuong pkdp

Sdn xuai khdng nguyen Sdi

Virus Edmonston Vcro7 da giam dgc tren 1 s6 loai li bao Vien Kitasato, mgt trong

so do la te bao Vero 7 lan tnigc khi chuyen sang Viet Nam, virut nay ti6p tyc dugc ciy truy6n 2 lin lien tiip tren tl bao Vero theo qui trinh san xuit khang nguyen cua vien Kitasato Nhtit ban ggi la chung Edmonston Vero 7/P2

Hdm luang khdng nguyen: 10''°CClD50/0,5ml

Tieu chuan tho sir dung: Khang the soi truac khi thii nghiem <4 don vi; Khoe manh,

khong bj bat ky benh ngoai da nao; Can nang: 2,0kg den 2,5 kg; Tho dugc each ly tu 7-10 ngay

Qui trinh gay ddp irng mien dich tren tho

Phucmg phap vien Kitasato-Nhat ban (Khang nguyen khong tinh che)

Stt

1

2

3

4

Ngay

0

07

14

28

Ngay thuc hien

10-09-2008

17-09-2008

24-09-2008

08-10-2008

Lay mau5 ml (Kiem tra khang the)

Lay mau Mo

Lay mau Ml (Lay mau giira ky)

Cach tiem

Tiem(l ) 2,5 + 2,5 (ml) (VRS+tadugc FCA) Tiem trong da nhieu vi tri, 0,1 ml/1 vi tri

Tiem (2) 5 (ml) VRS khong co chat phu gia

Tiem vao duoi da dui 2 ben, moi ben 2,5 ml Tiem (3) 5 (mi) VRS khong co chat phu gia

Tiem vao dual da dilii 2 ben, moi ben 2,5 ml Tiem (4) 10 (ml) VRS khong c6 chat phu gia

Tiem vao duoi da diii 2 ben,

1 Qui trinh san xuat huyet thanh soi

Nguyen vat tifu

Khang nguyen Soi tir chung Edmonston do vien Kitasato cung cip;

Tho trong lugng tir 1,8 ~ 2,5 kg;

Te bao Vero do vien Kitasato-Nhat ban cimg cip;

Moi trucmg hoa chit: MEM, NaHCOj 7,5%, BS, PBS (-), khang sinh P-S, Hank, chit bao quan vacxin, ta dugc tiem dap ling miin dich (Adjuvant): Ta dugc hoan chinh (CFA) va ta dugc khong hoan chlnh(IFA)

Vat lieu : Bom kim tiem trgn ta dugc, tu im CO2, pipet Aid, pipetman, diu tip cac loai, m4y ly tam lanh, ly tam thucmg, chai chira mlu (100 ml, 500 ml ), phiin 24 gilng Dung

cu lay mau tho,

Phutfttgphdp

Sdn xudt khdng nguyen Sdi

Virus Edmonston Vero7 da giam dgc tren 1 so loai tl bao Vien Kitasato, mot trong

so do \k te bao Vero 7 lin tmoc khi chuyin sang Viet Nam, virut nay tilp tuc dugc ciy

truyen 2 lan lien tilp tren tl bao Vero dieo qui trinh san xuit khang nguyen cua vien Kitasato Nhat ban ggi la chung Edmonston Vero 7/P2

Hdm lugng khdng nguyen: 10'°CClD50/0,5ml

Tieu chuin tho sir dung: Khang thi soi truoc khi thu nghiem <4 don vi; Khoe manh,

khong bi bit ky benh ngoai da nao; Can nang: 2,0kg din 2,5 kg; Tho dugc each ly tir 7-10 ngay

Qui trinh gay ddp ting mien dich tren tho

Phuong phap vien Kitasato-Nhat ban (Khang nguyen khong tinh chi)

Stt

1

2

3

4

Ngay

0

07

14

28

Ngay thirc hien

10-09-2008

17-09-2008

24 - 09 - 2008

08-10-2008

Lay rnau5 ml

(Kiem tia

khang the)

Lay mau Mo

Lay mau Ml (Lay mau giira ky)

Cach tiem

Tiem (1 ) 2,5 + 2,5 (ml) (VRS+tadugc FCA) Tiem trong da nhieu vj tri, 0,1 ml/1 vi tri

Tiem (2) 5 (ml) VRS khong co chat phu gia

Tiem vao ducyi da diii 2 ben, m6i ben 2,5 ml Tiem (3) 5 (ml) VRS khong co chat phu gia

Tiem vao duoi da diii 2 ben, moi ben 2,5 ml Tiem (4) 10 (ml) VRS khong co chat phu gia

Tiem vao duoi da ddi 2 ben,

5

6

42

49

22-10-2008

29 - 10 - 2008 (Lay het mau) Lay mau M2

chat phu gia Tiem vao tinh mach tai

Gay mien dich miii 1, tiem 2,5 ml khang nguyen + 2,5 ml ta dugc (ta dugc hoan toan), tiem trong da vimg lung Gay miin djch miii 2 sau tiem miii 1 la 7 ngay, tiem 5 ml khang nguyen, tiem duoi da diii Gay miin djch mOi 3 sau tiem miii 2 la 7 ngay, tiem 5 ml khang nguyen, tiem duoi da diii Gay mien dich mui 4 sau tiem mui 3 la 14 ngay, tiem 10

ml khang nguyen, tiem dual da diii Gay mien dich mui 5 sau tiem mui 4 la 14 ngay, tiem

10 ml khang nguyen, tiem tinh mach tai

Phuong phap cua CDC-Hoa ky (Khang nguyen tinh sach qua duone)

Stt

1

2

3

4

5

6

7

Ngay

0

14

21

28

35

49

Ngay thuc hien

10-09-2008

24 - 09 - 2008

01-10-2008

08-10-2008

15-10-2008

29-10-2008

Lay mau5 ml tinh mach tai (Kiem tra khang thi)

Lay mau Mo

Lay mau Ml

Lay mau M3 (Lay het mau)

Tiem

Tiem (1 ) MV + CFA (1ml / con , tiem trong da lung) Tiem (2 ) MV + IFA (1ml / con , tiem trong da lung) Tiem (3 ) MV + IFA (1ml / con, tiem trong da lung)

Liy mau kiem tra huyet thanh sgi 3 lan, lan 1 truoc khi gay mien dich mui 1; Lan 2 lay mau tai truoc khi gay miin dich miii 4 de kiem tra kha nang tao huyet thanh giita ky; Lan 3 liy mau toan bg sau 7 ngay gay mien dich mui 5 (ngay thii 49 ke tir ngay gay mien djch miii 1)

Tai ngay thir 49 liy toan bg mau tho vao chai det, bao quan 4°C qua dem , tach huyet thanh, ly tam 3000 vong / phut trong 20 phiit, lay huyit thanh, sau do bao quan - SO'C cho toi khi sir dung

Xac dinh hieu gia huyit thanh bang phucmg phap trung hoa tren tl bao vero mgt lop Huyit thanh dugc bit hoat 56°C trong 30 phut, sau do dugc pha loang bac 2 va dugc trung hoa vol 100 dcm vj khang nguyen soi, hin dich trung hoa dugc gay nhiim tren te bao vero

1 lop, sau 2 tuin dgc kit qua Hieu gia khang thi la nlng do huyit thanh pha loang nhit ma tji d6 tjo ra phan img trung hoa (tl bao binh thutmg khong bi huy hoai)

2 Qui trinh san xuat huyet thanh bai liet

Nguyen li(u va dung cu

Khang nguyen Bai liet typ I, II, III

Ta dugc (Freunds complete adjuvant - FCA, Freunds incomplete adjuvant -FIA) Th6:Khang thi soi targe khi thir nghiem <4 dcm vi; Khoe manh, khong bi bit ky benh ngoai da nao;Can nSng: 2,0kg din 2,5 kg; Tho dugc each ly tir 7-10 ngay

Te bao Hep2C cua to chiic Y te the giai

Xi lanh diing trgn khang nguyen va ta dugc

Ta dugc hoan chinh (CFA) va ta dugc khong hoan chinh(IFA)

- Moi trucmg MEM 10%, 2% FBS

- Trypsin Versen va mgt s6 dung djch khac

May ly tam

Phi&n nhua day det 96 gilng

Tu im 37°C va 36°C

- Pipet aid va pipetman cac loai

- Hood vo trimg va cac dyng cu khac phyc vu cho nghien cim

Phifffngphdp nghien cicu

Sdn xuit khdng nguyen bai lift: Nhan virut bai liet typ I, hoac typ II hoic typIII

tren te bao vero mgt lop vciri nong do 1 CCIDjo trong 300 tl bao vcri moi truang gay nhiim DMEM tai nhiet dg 37°C trong 72-96 gia, gat hin djch virut khi huy hoai tl bao

>90%

Tinh sifch khdng nguyen: Hon dich virut sau khi dugc dong tan 3 lan, ly tam 3000

vdng/phut trong 30 phut, lay nuac noi Can dugc sieu am va ly tam lay nuac noi Nuoc noi hgn lgi va dugc Igc voi co Igc lan lugt 0,65 (tm, 0,45 pm, 0,2 pm Hon djch virut dugc sieu Igc vgi co dac 100-200 lin va dugc sieu li tiim 36.000 v6ng/phut/3 gio tai 4'^C, lay can, hoa loang can bang BMS va bao quan tai -70''C cho den khi sir dung

- Cdch gdy ddp ling mien djch tgo khdng the bai li^t

Khdng nguySn: virut sabin typ 1 hoac typ 2 hoac typ 3

Him liryng khdng nguyen: 10'°CCID 50/0,1ml

Tho sir dyng: tieu chuin nhu tho sii dung cho san xuat huyet thanh scri

Cich gay dip irng mien djch: Phucmg phap CDC-Hoa Ky

1

2

3

4

5

6

7

0

14

28

42

56

70

84

Lay mau5 ml tinh

mach tai (Kiem tra

khang the)

Lay mau Mo

LiylnauMl

Lay mau M2

Lay mau M3

Lay mau M4

Lay mau M5

Lay mau M6

(Lay het mau)

Tiem (1) 0,2 + 0,2 (ml) (VRRT+la dugc FCA) Tiem trong da nhieu vi tri, 0,1 ml/1 vi tri Tiem (2) 1 + 1 (ml) (VRRT+ta dugc FIA) _ Tiem vao co diii 2 ben, moi ben 2 vj tri, moi vi ta' 0,5 ml

Tiem (3) 1 + 1 (ml) (VRRT+ta dugc FL\) Tiem vao co diii 2 ben, mil ben 2 vi tri, mli vi tri 0.5 ml

Tiem (4) 1 + 1 (ml) (VRRT+ta dugc FIA) Tiem vao ca diii 2 ben, moi ben 2 vi tri, moi vi ta' 0,5 ml

Tiem (5) 1 + 1 (ml) (VRRT+ta dugc FLA) Tiem vao co diii 2 ben, mli ben 2 vi tri, moi vi ta' 0,5 ml

Tiem (6) 1 + 1 (ml) (VRRT+ta dugc FIA) Tiem vao co diii 2 ben, moi ben 2 vi ta', moi vi ta' 0,5 ml

— _ _ _ ^

Thu huyet thanh : ly tam 3000 vong/phiit.l 0 phut Thu huyit thanh bao quan 6 -20°C Kiem tra hi^u gid: khang huyet thanh bang phucmg phap trung hoa vi lugng tren tl bao

Hep2C

3 Qui trinh san xuat huyet thanh rota

Nguyen vat U^u

Khang nguyen rota typ G1P8, G4P6, RRV

T^ bao than tho Rherus MA 104

Moi trucmg DMEM, 199

Tho tieu chuin

Ta dugc hoan chinh (CFA) va ta dugc khong hoan chinh(IFA)

Trypsin Versen va mgt s6 dung dich khac

May ly tam

- Phien nhua day det 96 giSng

- T u ^ 3 7 ' ' C va36''C

Pipet aid va pipetman cac loai

Hood vo trung va cac dung cu khac phuc vu cho nghien curu

Phuffng phap

Phifffngphdp sdn xudt khdng nguyen

Te bao MAI 04 mgt lap hoac te bao vero mgt lap, d?p dugc su dyng cho su nhan len

cua virut rota Te bao dugc rua 2 lan vcri moi truang DMEM im, sau do dugc dat tai 2il^C it

nhat 3 gia

Virut dugc hoat hoa vai trypsin nong do 15^1/ml tai nhiet dp 37°C trong 1 gia, sau

do hap phu virut len te bao tai nhiet do 37*'C trong 1 gia

Bo het moi truong gay nhiim, nia te bao 01 lan bang moi iruong DMEM am khong

CO trypsin, sau do cho them moi truang co trypsin vai nong do 10-20[ig/ml Dat chai te bao

da gay nhiim virut tai nhiet do 37''C trong 3 ngay

Gat h6n dich virut, dong tan 3 ISn, ly tam 3.000 v6ng/phut/30 phiit bo can Nuac n6i thu dugc sieu ly tam 25.000 v6ng/phut/2 gia tai 4'*C voi n6ng do ducmg 40% Bo nuac n6i, thu can va hoa can voi TNC, sau do pha loang khang nguyen thu dugc sau cho lugng virut khoang 10^-10^ ffii/ml

Phuvng phip gay dap irng mien dich tao khang the rota {CDC-Hoa Ky)

Stt Ngay Lay mau Cach tiem

Lay mau5 ml tinh

mach tai (Kiem tra

khang the)

Lay mau Mo Tiem (1 ) 0,2 + 0,2 (ml) (VRRT+ta dugc FCA) Tiem trone da nhiiu vi tri 0.1 ml/1 vi tri

2

3

4

5

6

7

14

28

42

56

70

84

LayTnauJvll

Lay mau M2

Lay mau M3

Lay mau M4

Lay mau M5

Lay mau M6

(Lay het mau)

Tiem vao ca dui 2 ben, mli ben 2 vi tri, mli vi tri 0,5

ml Tiem (3) 1 + 1 (ml) (VRRT+ta dugc FIA) _ Tiem vao eg did 2 ben, moi ben 2 vi tri, mli vi tri 0,5

ml Tiem (4) 1 + 1 (ml) (VRRT+ta dugc FIA) _ Tiem vao co did 2 ben, mli ben 2 vi tri, mli vi tri 0,5

ml Tiem (5)1 + 1 (ml) (VRRT+ta dugc FIA) _ Tiem vao ca dui 2 ben moi ben 2 vj tri, mli vj tri 0,5

ml Tiem (6) 1 + 1 (ml) (VRRT+ta dugc FIA) _ Tiem vao co diii 2 ben moi ben 2 vi tri, mli vi tri 0,5

ml

— _ _ _ _ ^

Thu huyet tiianh: ly tam 3000 v6ng/phut,10 phut Thu huyet thanh bao quan a <

-20 C cho toi khi sii dung

Kiem tra hi^u gid: khang the trung hoa bang phucmg phap ELIZA

4 Tieu chuan khang huyet thanh Soi

Tieu chi dinh gia

Vo trimg

Hi$u gia khang huydt

thanh

Tinh dac hieu

Tieu chuan Nhat Ban

Vo trimg

>4.000

Dac hieu bang thu nghiem nhan dang qua ky thuat miin dich huynh quang

Tieu chuin cff sir

Vo trimg

>4.000 cho thu nghiem xac dinh tac nhan ngo^ lai

> 1.000 cho thu nghiem nhan dang virut Soi

Dac hieu bSng thu nghiem nhan dang qua ky thuat mien dich huynh quang

5 Tieu chuan khang huyet thanh Bai liet Tieu chi danh gii

Vo trimg

Hi?u

gia

khang

huyet

thanh

bai

liet

Tinh dac

hi^u khang

huyet

thanh

Typ I

Typ II

Typ III

Typ I

Typ II

Typ III

Tieu chuin Nhat Ban

Vo trimg

>16.000

> 16.000

> 16.000

Cheo typ 2, 3 : <32

Cheo typ 2, 3 : <32

Cheo typ 2, 3 : <32

Tieu chuan ca so*

Vo trimg

>65.000 cho thit nghi?m xac dinh tac nhan ngoai lai

<65.000 cho thir nghi?m xac dinh hieu gia vac xin thanh pham

>8.000 cho thir nghiem xac dinh tac nhan ngoai lai

<8.000 cho thu nghiem xac dinh hieu gia vac xin thanh pham

>16.000 cho thir nghiem xic dinh tac nhan ngoai lai

<16.000 cho thir nghiem xac dinh hieu gia vac xin thanh phim Cheo typ 2, 3 : <32

Cheo voi typ 1,3: <16

Cheo vai typ 1 ,2 : <32

6 Tieu chuin khang huyet thanh Rota

Tieu chi dinh gii

Vo trimg

Tieu chuan Hoa Ky

Vo trimg

Tieu chuan ca sa

Vo trimg

khang

huyit

thanh

Tinh dac

hieu khang

huyet

thanh

Wa

DSl

RRV

RRVmix

G1P8

Wa

DSl

RRV

RRVmix

5.000 5.000

Dac hieu bang thir nghiem nhan dang qua ky thuat miin dich huynh quang

>100.000

> 1.000.000

> 100.000

Dac hieu bang thu nghiem nhan dang qua ky thuat mien dich huynh quang

7 Bai bao da dang Bit bao da dang: 01 bai

Nghien cim san xult huyit thanh Soi sir dung cho kiim dinh vic xin Tap Chi Y hgc

du phong, 2008, Tap XVIII si 6(98), tr.74-78

8 Dao tao Thac sy

Bko t^o: 01 thac sS'

Ten dl tai: Nghien ciiu san xult khang huyet thanh Bai liet tren tho va danh gia tinh an toan cua vac xin Bai liet trong phong thi nghiem

Chuyen nhanh: Thii y

Ma si 60.62.50

Ba bao ve vol diem xult sac nam 2008